Antibodies that Bind Colony Stimulating Factor 1 Receptor (CSF1R)

ABSTRACT

Antibodies that bind CSF1R are provided. Antibody heavy chains and light chains that are capable of forming antibodies that bind CSF1R are also provided. Polynucleotides encoding antibodies to CSF1R are provided. Polynucleotides encoding antibody heavy chains and lights chains are also provided. Methods of treatment using antibodies to CSF1R are provided. Such methods include, but are not limited to, methods of treating rheumatoid arthritis, bone loss, and multiple sclerosis.

This application is a divisional of U.S. patent application Ser. No.16/737,217, filed Jan. 8, 2020, which is a divisional of U.S. patentapplication Ser. No. 15/935,621, filed Mar. 26, 2018, now U.S. Pat. No.10,562,970, which is a divisional of U.S. patent application Ser. No.15/279,853 filed Sep. 29, 2016, now U.S. Pat. No. 9,957,327, which is adivisional of U.S. patent application Ser. No. 14/924,568, filed Oct.27, 2015, now U.S. Pat. No. 9,695,243, which is a divisional of U.S.patent application Ser. No. 14/266,209, filed Apr. 30, 2014, now U.S.Pat. No. 9,200,075, which is a divisional of U.S. patent applicationSer. No. 13/464,503, filed May 4, 2012, now U.S. Pat. No. 8,747,845,which is a divisional of U.S. patent application Ser. No. 13/100,990,filed May 4, 2011, now U.S. Pat. No. 8,206,715, which claims the benefitof U.S. Provisional Application No. 61/331,177, filed May 4, 2010, eachof which is incorporated by reference herein in its entirety for anypurpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2015-10-26_01134-0014-03US_SeqList_ST25.txt” created on Oct. 26, 2015,which is 147,416 bytes in size. The information in the electronic formatof the sequence listing is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

Antibodies that bind CSF1R are provided. Antibody heavy chains and lightchains that are capable of forming antibodies that bind CSF1R are alsoprovided. In addition, antibodies, heavy chains, and light chainscomprising one or more particular complementarity determining regions(CDRs) are provided. Polynucleotides encoding antibodies to CSF1R areprovided. Polynucleotides encoding antibody heavy chains or lightschains are also provided. Methods of treatment using antibodies to CSF1Rare provided. Such methods include, but are not limited to, methods oftreating rheumatoid arthritis, bone loss, and multiple sclerosis.

BACKGROUND

Colony stimulating factor 1 receptor (referred to herein as CSF1R; alsoreferred to in the art as FMS, FIM2, C-FMS, and CD115) is a single-passtransmembrane receptor with an N-terminal extracellular domain (ECD) anda C-terminal intracellular domain with tyrosine kinase activity. Ligandbinding of CSF1 or the interleukin 34 ligand (referred to herein asIL34; Lin et al., Science 320: 807-11 (2008)) to CSF1R leads to receptordimerization, upregulation of CSF1R protein tyrosine kinase activity,phosphorylation of CSF1R tyrosine residues, and downstream signalingevents. Both CSF1 and IL34 stimulate monocyte survival, proliferation,and differentiation into macrophages.

Many tumor cells have been found to secrete CSF1, which activatesmonocyte/macrophage cells through CSF1R. The level of CSF1 in tumors hasbeen shown to correlate with the level of tumor-associated macrophages(TAMs) in the tumor. Higher levels of TAMs have been found to correlatewith poorer patient prognoses. In addition, CSF1 has been found topromote tumor growth and progression to metastasis in, for example,human breast cancer xenografts in mice. See, e.g., Paulus et al., CancerRes. 66: 4349-56 (2006). Further, CSF1R appears to play a role inosteolytic bone destruction in bone metastasis, as a small moleculeinhibitor of receptor tyrosine kinase activity suppresses thatdestruction. See, e.g., Ohno et al., Mol. Cancer Ther. 5: 2634-43(2006).

CSF1 and its receptor have also been found to be involved in variousinflammatory and autoimmune diseases. See, e.g., Hamilton, Nat. Rev. 8:533-44 (2008). For example, synovial endothelial cells from jointsafflicted with rheumatoid arthritis have been found to produce CSF1,suggesting a role for CSF1 and its receptor in the disease. BlockingCSF1R activity with an antibody results in positive clinical effects inmouse models of arthritis, including a reduction in the destruction ofbone and cartilage and a reduction in macrophage numbers. See, e.g.,Kitaura et al., J. Clin. Invest. 115: 3418-3427 (2005).

Mature differentiated myeloid lineage cells such as macrophages,microglial cells, and osteoclasts contribute to pathology of variousdiseases such as rheumatoid arthritis, multiple sclerosis and diseasesof bone loss. Differentiated myeloid lineage cells are derived fromperipheral blood monocyte intermediates. CSF1R stimulation contributesto development of monocytes from bone marrow precursors, to monocyteproliferation and survival, and to differentiation of peripheral bloodmonocytes into differentiated myeloid lineage cells such as macrophages,microglial cells, and osteoclasts. CSF1R stimulation thus contributes toproliferation, survival, activation, and maturation of differentiatedmyeloid lineage cells, and in the pathologic setting, CSF1R stimulationcontributes to the ability of differentiated myeloid lineage cells tomediate disease pathology.

Additional antagonists of CSF1R signaling would therefore be useful inthe treatment of various CSF1R-related diseases, such as cancer,inflammatory conditions, and autoimmune diseases.

SUMMARY

The present inventors have invented a new set of antibodies, includinghumanized antibodies, directed against human CSF1R extracellular domain(CSF1R ECD). A Fab phage display library was made from spleens of micethat were immunized with a human CSF1R ECD-Fc fusion protein. 1056 phageclones expressing Fabs that bind to CSF-1R ECD-Fc were isolated throughpanning of this library. When the 1056 Fabs were expressed as purifiedprotein, 668 were found to bind to CSF1R ECD. Of those 668 binding Fabs,only 121 Fabs blocked binding of CSF1 and/or IL34 to CSF1R. Only 33 ofthose Fabs were found to block binding of both CSF1 and IL34 to CSF1R.Upon sequencing, the 33 Fabs represented 19 unique sets of sequences.Eleven Fabs with subnanomolar affinity for human CSF1R ECD were chosento make chimeric antibodies for further study. Based on the human andcynomolgus monkey CSF1R binding affinities, blocking of CSF1 and IL34binding to CSF1R, and inhibition of ligand-induced phorphorylation ofCSF1R, three chimeric antibodies were selected for humanization, andsixteen humanized antibodies were made based on those three chimericantibodies.

Fourteen of the sixteen humanized antibodies retained subnanomolarbinding affinities for human CSF1R ECD. See, e.g., Table 5. Thesehumanized antibodies block binding of both ligands CSF1 and IL34 tohuman CSF1R, and many also block binding of both CSF1 and IL34 tocynomolgus monkey CSF1R. See, e.g., Table 4.

For therapeutic drug development, it is beneficial to have antibodiesthat bind to both human and cynomolgus monkey antigens with similaraffinity. The three chimeric antibodies chosen for humanization wereselected in part because they had similar binding affinities for humanand cynomolgus CSF1R ECD. Most of the humanized versions of one of thechimeric antibodies, 0302, however, lost significant binding affinityfor cynomolgus monkey CSF1R ECD upon humanization, although theyretained strong human CSF1R ECD binding affinity. See, e.g., Table 3.Humanized versions of 0301 and 0311 retained similarly strong binding toboth human and cynomolgus monkey CSF1R ECD, with binding affinitydifferences for the two species of less than about 2-fold.

Based on CSF1R binding affinities, ligand inhibition, and the potentialfor immunogenicity, three humanized antibodies were selected foradditional studies. The three humanized antibodies were derived from thetwo chimeric antibodies that did not significantly lose cynomolgusmonkey CSF1R binding affinity upon humanization. Those three humanizedantibodies inhibit ligand-induced phosphorylation of human CSF1R, andalso block ligand-induced proliferation and survival responses inprimary human monocytes. See, e.g., Tables 6 and 7, and FIGS. 10 and 11.Thus, these antibodies are useful for treating diseases involving, forexample, ligand-induced proliferation and survival responses in primaryhuman monocytes.

Blocking CSF1R-induced responses with an anti-CSF1R antibody shouldinhibit proliferation, survival, activation, maturation ofdifferentiated myeloid lineage cells and attenuate their ability tomediate disease pathology. In addition, blocking CSF1R-induced responseswith an anti-CSF1R antibody should inhibit differentiation of peripheralblood monocyte intermediates into differentiated myeloid lineage cells,decreasing the number of pathology-mediating differentiated myeloidlineage cells.

Accordingly, the humanized anti-CSF1R antibodies described herein can beused to treat chronic diseases with extant symptoms by inhibiting theability of differentiated myeloid lineage cells to mediate diseasepathology. The humanized antibodies can also be used to treat chronicdiseases that are relapsing and remitting in nature by inhibiting thedevelopment of new pathology-mediating myeloid lineage cellsdifferentiated from peripheral blood monocytes during the remittingphase of the disease, thus attenuating the number of and new formationof the pathology-mediating cells.

In some embodiments, an isolated antibody comprising a heavy chain and alight chain is provided, wherein the antibody binds to CSF1R. In someembodiments, the heavy chain and/or light chain have the followingstructure.

In some embodiments, the heavy chain comprises a sequence that is atleast 90%, at least 95%, at least 97%, at least 99%, or 100% identicalto a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45. In someembodiments, the light chain comprises a sequence that is at least 90%,at least 95%, at least 97%, at least 99%, or 100% identical to asequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52. In someembodiments, the heavy chain comprises a sequence that is at least 90%,at least 95%, at least 97%, at least 99%, or 100% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, and thelight chain comprises a sequence that is at least 90%, at least 95%, atleast 97%, at least 99%, or 100% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52.

In some embodiments, the HC CDR1, HC CDR2, and HC CDR3 comprise a set ofsequences selected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs:21, 22, and 23; and (c) SEQ ID NOs: 27, 28, and 29. In some embodiments,the LC CDR1, LC CDR2, and LC CDR3 comprise a set of sequences selectedfrom: (a) SEQ ID NOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26;and (c) SEQ ID NOs: 30, 31, and 32.

In some embodiments, the heavy chain comprises an HC CDR1, HC CDR2, andHC CDR3, wherein the HC CDR1, HC CDR2, and HC CDR3 comprise a set ofsequences selected from: (a) SEQ ID NOs: 15, 16, and 17; (b) SEQ ID NOs:21, 22, and 23; and (c) SEQ ID NOs: 27, 28, and 29; and the light chaincomprises an LC CDR1, LC CDR2, and LC CDR3, wherein the LC CDR1, LCCDR2, and LC CDR3 comprise a set of sequences selected from: (a) SEQ IDNOs: 18, 19, and 20; (b) SEQ ID NOs: 24, 25, and 26; and (c) SEQ ID NOs:30, 31, and 32.

In some embodiments, an isolated antibody is provided, wherein theantibody comprises a heavy chain and a light chain, wherein the antibodycomprises: (a) a heavy chain comprising a sequence that is at least 95%,at least 97%, at least 99%, or 100% identical to SEQ ID NO: 9 and alight chain comprising a sequence that is at least 95%, at least 97%, atleast 99%, or 100% identical to SEQ ID NO: 10; (b) a heavy chaincomprising a sequence that is at least 95%, at least 97%, at least 99%,or 100% identical to SEQ ID NO: 11 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 12; (c) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 13 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 14; (d) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 39 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 46; (e) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 40 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 46; (f) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 41 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 46; (g) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 39 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 47; (h) a heavychain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 40 and a light chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 47; (i) a heavy chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 41 and a light chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 47; and (j) aheavy chain comprising a sequence that is at least 95%, at least 97%, atleast 99%, or 100% identical to SEQ ID NO: 42 and a light chaincomprising a sequence that is at least 95%, at least 97%, at least 99%,or 100% identical to SEQ ID NO: 48; (k) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 42 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 49; (l) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 42 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 50; (m) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 43 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 48; (n) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 43 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 49; (o) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 43 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 50; (p) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 44 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 51; (q) a heavy chain comprising asequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 44 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 52; (r) a heavy chain comprising a sequence that is at least 95%, atleast 97%, at least 99%, or 100% identical to SEQ ID NO: 45 and a lightchain comprising a sequence that is at least 95%, at least 97%, at least99%, or 100% identical to SEQ ID NO: 51; or (s) a heavy chain comprisinga sequence that is at least 95%, at least 97%, at least 99%, or 100%identical to SEQ ID NO: 45 and a light chain comprising a sequence thatis at least 95%, at least 97%, at least 99%, or 100% identical to SEQ IDNO: 52.

In some embodiments, an antibody is provided, wherein the antibodycomprises a heavy chain and a light chain, wherein the antibodycomprises: (a) a heavy chain comprising a heavy chain (HC) CDR1 havingthe sequence of SEQ ID NO: 15, an HC CDR2 having the sequence of SEQ IDNO: 16, and an HC CDR3 having the sequence of SEQ ID NO: 17, and a lightchain comprising a light chain (LC) CDR1 having the sequence of SEQ IDNO: 18, a LC CDR2 having the sequence of SEQ ID NO: 19, and a LC CDR3having the sequence of SEQ ID NO: 20; (b) a heavy chain comprising aheavy chain (HC) CDR1 having the sequence of SEQ ID NO: 21, an HC CDR2having the sequence of SEQ ID NO: 22, and an HC CDR3 having the sequenceof SEQ ID NO: 23, and a light chain comprising a light chain (LC) CDR1having the sequence of SEQ ID NO: 24, a LC CDR2 having the sequence ofSEQ ID NO: 25, and a LC CDR3 having the sequence of SEQ ID NO: 26; or(c) a heavy chain comprising a heavy chain (HC) CDR1 having the sequenceof SEQ ID NO: 27, an HC CDR2 having the sequence of SEQ ID NO: 28, andan HC CDR3 having the sequence of SEQ ID NO: 29, and a light chaincomprising a light chain (LC) CDR1 having the sequence of SEQ ID NO: 30,a LC CDR2 having the sequence of SEQ ID NO: 31, and a LC CDR3 having thesequence of SEQ ID NO: 32.

In some embodiments, an antibody comprises a heavy chain and a lightchain, wherein the antibody comprises: (a) a heavy chain comprising asequence of SEQ ID NO: 53 and a light chain comprising a sequence of SEQID NO: 60; (b) a heavy chain comprising a sequence of SEQ ID NO: 53 anda light chain comprising a sequence of SEQ ID NO: 61; or (c) a heavychain comprising a sequence of SEQ ID NO: 58 and a light chaincomprising a sequence of SEQ ID NO: 65. In some embodiments, an antibodycomprises a heavy chain and a light chain, wherein the antibodycomprises: (a) a heavy chain consisting of the sequence of SEQ ID NO: 53and a light chain consisting of the sequence of SEQ ID NO: 60; (b) aheavy chain consisting of the sequence of SEQ ID NO: 53 and a lightchain consisting of the sequence of SEQ ID NO: 61; or (c) a heavy chainconsisting of the sequence of SEQ ID NO: 58 and a light chain consistingof the sequence of SEQ ID NO: 65.

In some embodiments, an antibody is a humanized antibody. In someembodiments, an antibody is selected from a Fab, an Fv, an scFv, a Fab′,and a (Fab′)₂. In some embodiments, an antibody is a chimeric antibody.In some embodiments, an antibody is selected from an IgA, an IgG, and anIgD. In some embodiments, an antibody is an IgG. In some embodiments, anantibody is an IgG4. In some embodiments, an antibody is an IgG4comprising an S241P mutation in at least one IgG4 heavy chain constantregion.

In some embodiments, an antibody binds to human CSF1R and/or binds tocynomolgus CSF1R. In some embodiments, an antibody blocks ligand bindingto CSF1R. In some embodiments, an antibody blocks binding of CSF1 and/orIL34 to CSF1R. In some embodiments, an antibody inhibits ligand-inducedCSF1R phosphorylation. In some embodiments, an antibody inhibits CSF1-and/or IL34-induced CSF1R phosphorylation. In some embodiments, anantibody binds to human CSF1R with an affinity (K_(D)) of less than 1nM. In some embodiments, antibody inhibits monocyte proliferation and/orsurvival responses in the presence of CSF1 or IL34.

In some embodiments, a pharmaceutical composition comprising an antibodythat binds CSF1R is provided.

In some embodiments, an isolated nucleic acid is provided, wherein theisolated nucleic acid comprises a polynucleotide sequence that encodes aheavy chain described above. In some embodiments, an isolated nucleicacid encodes a light chain described above. In some embodiments, anisolated nucleic acid encodes a heavy chain described above and a lightchain described above. In some embodiments, a composition is provided,wherein the composition comprises a first nucleic acid that comprises apolynucleotide sequence that encodes a heavy chain described above, anda second nucleic acid that comprises a polynucleotide sequence thatencodes a light chain described above. In some embodiments, a host cellcomprising a nucleic acid or a composition described above is provided.In some embodiments, a host cell is a eukaryotic host cell. In someembodiments, a host cell is a mammalian host cell. In some embodiments,a host cell is selected from a CHO cell, a 293 cell, an NSO cell, and aPER.C6 cell. In some embodiments, a host cell is a 293-6E cell or a DG44cell.

In some embodiments, methods of treating disease comprisingadministering to a patient a pharmaceutical composition comprising anantibody that binds CSF1R is provided. In some embodiments, a method oftreating multiple sclerosis comprising administering to a patient apharmaceutical composition comprising an antibody that binds CSF1R isprovided. In some embodiments, a method of treating rheumatoid arthritiscomprising administering to a patient a pharmaceutical compositioncomprising an antibody that binds CSF1R is provided. In someembodiments, a method of treating osteolytic bone loss comprisingadministering to a patient a pharmaceutical composition comprising anantibody that binds CSF1R is provided. In some embodiments, theosteolytic bone loss is selected from osteoporosis, metastasis-inducedosteolytic bone loss, and rheumatoid arthritis-induced bone loss. Insome embodiments, a method of treating cancer comprising administeringto a patient a pharmaceutical composition comprising an antibody thatbinds CSF1R is provided. In some embodiments, the cancer is selectedfrom breast cancer, prostate cancer, endometrial cancer, bladder cancer,kidney cancer, esophageal cancer, squamous cell carcinoma, uvealmelanoma, follicular lymphoma, renal cell carcinoma, cervical cancer,ovarian cancer, lung cancer, colorectal cancer, brain cancer, pancreaticcancer, head and neck cancer, liver cancer, leukemia, lymphoma,Hodgkin's disease, multiple myeloma, melanoma, astrocytoma, stomachcancer, and pulmonary adenocarcinoma.

In some embodiments, a method of treating an inflammatory conditioncomprising administering to a patient a pharmaceutical compositioncomprising an antibody that binds CSF1R is provided.

In some embodiments, antibodies that bind CSF1R and compositionscomprising antibodies that bind CSF1R are provided for use in methods oftreatment of human or animals. In some embodiments, antibodies that bindCSF1R and compositions comprising antibodies that bind CSF1R areprovided for use in a method of treating rheumatoid arthritis in a humanor animal. In some embodiments, antibodies that bind CSF1R andcompositions comprising antibodies that bind CSF1R are provided for usein a method of treating multiple sclerosis in a human or animal. In someembodiments, antibodies that bind CSF1R and compositions comprisingantibodies that bind CSF1R are provided for use in a method of treatingcancer in a human or animal. In some embodiments, antibodies that bindCSF1R and compositions comprising antibodies that bind CSF1R areprovided for use in a method of treating an inflammatory condition in ahuman or animal.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show an alignment of the humanized heavy chain variableregions for each of humanized antibodies Ab1 to Ab16, as discussed inExample 4. Boxed residues are amino acids in the human acceptor sequencethat were changed back to the corresponding mouse residue.

FIGS. 2A-C show an alignment of the humanized light chain variableregions for each of humanized antibodies Ab1 to Ab16, as discussed inExample 4 Boxed amino acids are residues in the human acceptor sequencethat were changed back to the corresponding mouse residue.

FIGS. 3A-C show binding curves for certain humanized antibodies bindingto human CSF1R ECD, as described in Example 5. FIG. 3A shows bindingcurves for parental chimeric antibodies (cAb) 0301 and humanizedantibodies (huAb) 0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6(h0301-L0H0, h0301-L0H1, h0301-L0H2, h0301-L1H0, h0301-L1H1, andh0301-L1H2, respectively). FIG. 3B shows binding curves for parental cAb0302 and humanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4,0302.5, and 0302.6 (h0302-L0H1, h0302-L1H1, h0302-L2H1, h0302-L0H2,h0302-L1H2, and h0302-L2H2, respectively). FIG. 3C shows binding curvesfor parental cAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2,0311.3, and 0311.4 (h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2,respectively).

FIGS. 4A-C show binding curves for certain humanized antibodies bindingto cynomolgus CSF1R ECD, as described in Example 5. FIG. 4A showsbinding curves for parental cAb 0301 and humanized antibodies (huAb)0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (h0301-L0H0,h0301-L0H1, h0301-L0H2, h0301-L1H0, h0301-L1H1, and h0301-L1H2,respectively). FIG. 4B shows binding curves for parental cAb 0302 andhumanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and0302.6 (h0302-L0H1, h0302-L1H1, h0302-L2H1, h0302-L0H2, h0302-L1H2, andh0302-L2H2, respectively). FIG. 4C shows binding curves for parental cAb0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and 0311.4(h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2, respectively).

FIGS. 5A-C show binding curves for certain humanized antibodies bindingto mouse CSF1R ECD, as described in Example 5. FIG. 5A shows bindingcurves for parental cAb 0301 and humanized antibodies (huAb) 0301.1,0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (h0301-L0H0, h0301-L0H1,h0301-L0H2, h0301-L1H0, h0301-L1H1, and h0301-L1H2, respectively). FIG.5B shows binding curves for parental cAb 0302 and humanized antibodies(huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and 0302.6 (h0302-L0H1,h0302-L1H1, h0302-L2H1, h0302-L0H2, h0302-L1H2, and h0302-L2H2,respectively). FIG. 5C shows binding curves for parental cAb 0311 andhumanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and 0311.4(h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2, respectively).

FIGS. 6A-C show inhibition of CSF1 induced CSF1R phosphorylation bycertain humanized antibodies, as described in Example 6. FIG. 6A showsblocking curves for parental cAb 0301 and humanized antibodies (huAb)0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (h0301-L0H0,h0301-L0H1, h0301-L0H2, h0301-L1H0, h0301-L1H1, and h0301-L1H2,respectively). FIG. 6B shows blocking curves for parental cAb 0302 andhumanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and0302.6 (h0302-L0H1, h0302-L1H1, h0302-L2H1, h0302-L0H2, h0302-L1H2, andh0302-L2H2, respectively). FIG. 6C shows blocking curves for parentalcAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and0311.4 (h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2,respectively).

FIGS. 7A-C show inhibition of IL34 induced CSF1R phosphorylation bycertain humanized antibodies, as described in Example 6. FIG. 7A showsblocking curves for parental cAb 0301 and humanized antibodies (huAb)0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (h0301-L0H0,h0301-L0H1, h0301-L0H2, h0301-L1H0, h0301-L1H1, and h0301-L1H2,respectively). FIG. 7B shows blocking curves for parental cAb 0302 andhumanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and0302.6 (h0302-L0H1, h0302-L1H1, h0302-L2H1, h0302-L0H2, h0302-L1H2, andh0302-L2H2, respectively). FIG. 7C shows blocking curves for parentalcAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and0311.4 (h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2,respectively).

FIGS. 8A-C show blocking of human CSF1 binding to cynomolgus CSF1R ECDby certain humanized antibodies, as described in Example 7. FIG. 8Ashows blocking curves for parental cAb 0301 and humanized antibodies(huAb) 0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (h0301-L0H0,h0301-L0H1, h0301-L0H2, h0301-L1H0, h0301-L1H1, and h0301-L1H2,respectively). FIG. 8B shows blocking curves for parental cAb 0302 andhumanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and0302.6 (h0302-L0H1, h0302-L1H1, h0302-L2H1, h0302-L0H2, h0302-L1H2, andh0302-L2H2, respectively). FIG. 8C shows blocking curves for parentalcAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and0311.4 (h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2,respectively).

FIGS. 9A-C show blocking of human IL34 binding to cynomolgus CSF1R ECDby certain humanized antibodies, as described in Example 7. FIG. 9Ashows blocking curves for parental cAb 0301 and humanized antibodies(huAb) 0301.1, 0301.2, 0302.3, 0301.4, 0301.5, and 0301.6 (h0301-L0H0,h0301-L0H1, h0301-L0H2, h0301-L1H0, h0301-L1H1, and h0301-L1H2,respectively). FIG. 9B shows blocking curves for parental cAb 0302 andhumanized antibodies (huAb) 0302.1, 0302.2, 0302.3, 0302.4, 0302.5, and0302.6 (h0302-L0H1, h0302-L1H1, h0302-L2H1, h0302-L0H2, h0302-L1H2, andh0302-L2H2, respectively). FIG. 9C shows blocking curves for parentalcAb 0311 and humanized antibodies (huAb) 0311.1, 0311.2, 0311.3, and0311.4 (h0311-L0H1, h0311-L1H1, h0311-L0H2, and h0311-L1H2,respectively).

FIGS. 10A and B show blocking of CSF1- (10A) and IL34- (10B) inducedCSF1R phosphorylation in CHO cells expressing human CSF1R by humanizedantibodies 0301-L0H0, 0301-L1H0, and 0311-L0H1, as described in Example9.

FIGS. 11A and B show blocking of CSF1- (11A) and IL34- (11B) inducedmonocyte proliferation/survival responses by humanized antibodies0301-L0H0, 0301-L1H0, and 0311-L0H1, as described in Example 10.

FIGS. 12A-C show that humanized antibodies 0301-L0H0, 0301-L1H0, and0311-L0H1 do not stimulate primary monocyte proliferation or survival,using monocytes from three different donors, as described in Example 11.

DETAILED DESCRIPTION

Methods of treating diseases comprising administering novel antibodiesto CSF1R are provided. All of the antibodies have binding affinities forhuman CSF1R ECD of less than 2 nM, and all but two of the humanizedantibodies have sub-nanomolar binding affinities for human CSF1R ECD.Further, the new antibodies block binding of both CSF1 and IL34 to humanCSF1R, and inhibit ligand-induced phosphorylation of human CSF1R. Manyof the new antibodies also block binding of CSF1 and IL34 to cynomolgusCSF1R, which facilitates in vivo experiments to support the developmentof anti-CSF1R antibody therapeutics. The new antibodies are thereforewell suited for therapeutic use in human diseases, including, but notlimited to, cancer, autoimmune diseases, and inflammatory conditions.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Exemplary techniques used in connection with recombinant DNA,oligonucleotide synthesis, tissue culture and transformation (e.g.,electroporation, lipofection), enzymatic reactions, and purificationtechniques are known in the art. Many such techniques and procedures aredescribed, e.g., in Sambrook et al. Molecular Cloning: A LaboratoryManual (2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)), among other places. In addition, exemplarytechniques for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients arealso known in the art.

In this application, the use of “or” means “and/or” unless statedotherwise. In the context of a multiple dependent claim, the use of “or”refers back to more than one preceding independent or dependent claim inthe alternative only. Also, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The terms “nucleic acid molecule” and “polynucleotide” may be usedinterchangeably, and refer to a polymer of nucleotides. Such polymers ofnucleotides may contain natural and/or non-natural nucleotides, andinclude, but are not limited to, DNA, RNA, and PNA. “Nucleic acidsequence” refers to the linear sequence of nucleotides that comprise thenucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

The term “CSF1R” refers herein to the full-length CSF1R, which includesthe N-terminal ECD, the transmembrane domain, and the intracellulartyrosine kinase domain, with or without an N-terminal leader sequence.In some embodiments, the CSF1R is a human CSF1R having the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The term “CSF1R extracellular domain” (“CSF1R ECD”) as used hereinrefers to a CSF1R polypeptide that lacks the intracellular andtransmembrane domains. CSF1R ECDs include the full-length CSF1R ECD andCSF1R ECD fragments that are capable of binding CSF1R and/or IL34. Thehuman full-length CSF1R ECD is defined herein as comprising either aminoacids 1 to 512 (i.e., including the leader sequence) or amino acids 20to 512 (i.e., lacking the leader sequence) of SEQ ID NO: 2. In someembodiments, a human CSF1R ECD fragment comprises amino acids 20 to 506of SEQ ID NO: 2 (see SEQ ID NO:5). In some embodiments, a human CSF1Rfragment ends at amino acid 507, 508, 509, 510, or 511. In someembodiments, a cynoCSF1R ECD comprises the sequence of SEQ ID NO: 7(with leader sequence) or amino acids 20 to 506 of SEQ ID NO: 7 (withoutleader sequence).

The term “antibody” as used herein refers to a molecule comprising atleast complementarity-determining region (CDR) 1, CDR2, and CDR3 of aheavy chain and at least CDR1, CDR2, and CDR3 of a light chain, whereinthe molecule is capable of binding to antigen. The term antibodyincludes, but is not limited to, fragments that are capable of bindingantigen, such as Fv, single-chain Fv (scFv), Fab, Fab′, and (Fab′)₂. Theterm antibody also includes, but is not limited to, chimeric antibodies,humanized antibodies, and antibodies of various species such as mouse,human, cynomolgus monkey, etc.

In some embodiments, an antibody comprises a heavy chain variable regionand a light chain variable region. In some embodiments, an antibodycomprises at least one heavy chain comprising a heavy chain variableregion and at least a portion of a heavy chain constant region, and atleast one light chain comprising a light chain variable region and atleast a portion of a light chain constant region. In some embodiments,an antibody comprises two heavy chains, wherein each heavy chaincomprises a heavy chain variable region and at least a portion of aheavy chain constant region, and two light chains, wherein each lightchain comprises a light chain variable region and at least a portion ofa light chain constant region. As used herein, a single-chain Fv (scFv),or any other antibody that comprises, for example, a single polypeptidechain comprising all six CDRs (three heavy chain CDRs and three lightchain CDRs) is considered to have a heavy chain and a light chain. Insome such embodiments, the heavy chain is the region of the antibodythat comprises the three heavy chain CDRs and the light chain in theregion of the antibody that comprises the three light chain CDRs.

The term “heavy chain variable region” as used herein refers to a regioncomprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. Insome embodiments, a heavy chain variable region also comprises at leasta portion of an FR1 and/or at least a portion of an FR4. In someembodiments, a heavy chain CDR1 corresponds to Kabat residues 26 to 35;a heavy chain CDR2 corresponds to Kabat residues 50 to 65; and a heavychain CDR3 corresponds to Kabat residues 95 to 102. See, e.g., KabatSequences of Proteins of Immunological Interest (1987 and 1991, NIH,Bethesda, Md.); and FIG. 1. In some embodiments, a heavy chain CDR1corresponds to Kabat residues 31 to 35; a heavy chain CDR2 correspondsto Kabat residues 50 to 65; and a heavy chain CDR3 corresponds to Kabatresidues 95 to 102. See id.

The term “heavy chain constant region” as used herein refers to a regioncomprising at least three heavy chain constant domains, C_(H)1, C_(H)2,and C_(H)3. Nonlimiting exemplary heavy chain constant regions includeγ, δ, and α. Nonlimiting exemplary heavy chain constant regions alsoinclude ε and μ. Each heavy constant region corresponds to an antibodyisotype. For example, an antibody comprising a γ constant region is anIgG antibody, an antibody comprising a δ constant region is an IgDantibody, and an antibody comprising an α constant region is an IgAantibody. Further, an antibody comprising a μ constant region is an IgMantibody, and an antibody comprising an F constant region is an IgEantibody. Certain isotypes can be further subdivided into subclasses.For example, IgG antibodies include, but are not limited to, IgG1(comprising a γ₁ constant region), IgG2 (comprising a γ₂ constantregion), IgG3 (comprising a γ₃ constant region), and IgG4 (comprising aγ₄ constant region) antibodies; IgA antibodies include, but are notlimited to, IgA1 (comprising an α₁ constant region) and IgA2 (comprisingan α₂ constant region) antibodies; and IgM antibodies include, but arenot limited to, IgM1 and IgM2.

In some embodiments, a heavy chain constant region comprises one or moremutations (or substitutions), additions, or deletions that confer adesired characteristic on the antibody. A nonlimiting exemplary mutationis the S241P mutation in the IgG4 hinge region (between constant domainsC_(H)1 and C_(H)2), which alters the IgG4 motif CPSCP to CPPCP, which issimilar to the corresponding motif in IgG1. That mutation, in someembodiments, results in a more stable IgG4 antibody. See, e.g., Angal etal., Mol. Immunol. 30: 105-108 (1993); Bloom et al., Prot. Sci. 6:407-415 (1997); Schuurman et al., Mol. Immunol. 38: 1-8 (2001).

The term “heavy chain” as used herein refers to a polypeptide comprisingat least a heavy chain variable region, with or without a leadersequence. In some embodiments, a heavy chain comprises at least aportion of a heavy chain constant region. The term “full-length heavychain” as used herein refers to a polypeptide comprising a heavy chainvariable region and a heavy chain constant region, with or without aleader sequence.

The term “light chain variable region” as used herein refers to a regioncomprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. Insome embodiments, a light chain variable region also comprises an FR1and/or an FR4. In some embodiments, a light chain CDR1 corresponds toKabat residues 24 to 34; a light chain CDR2 corresponds to Kabatresidues 50 to 56; and a light chain CDR3 corresponds to Kabat residues89 to 97. See, e.g., Kabat Sequences of Proteins of ImmunologicalInterest (1987 and 1991, NIH, Bethesda, Md.); and FIG. 1.

The term “light chain constant region” as used herein refers to a regioncomprising a light chain constant domain, C_(L). Nonlimiting exemplarylight chain constant regions include λ and κ.

The term “light chain” as used herein refers to a polypeptide comprisingat least a light chain variable region, with or without a leadersequence. In some embodiments, a light chain comprises at least aportion of a light chain constant region. The term “full-length lightchain” as used herein refers to a polypeptide comprising a light chainvariable region and a light chain constant region, with or without aleader sequence.

A “chimeric antibody” as used herein refers to an antibody comprising atleast one variable region from a first species (such as mouse, rat,cynomolgus monkey, etc.) and at least one constant region from a secondspecies (such as human, cynomolgus monkey, etc.). In some embodiments, achimeric antibody comprises at least one mouse variable region and atleast one human constant region. In some embodiments, a chimericantibody comprises at least one cynomolgus variable region and at leastone human constant region. In some embodiments, all of the variableregions of a chimeric antibody are from a first species and all of theconstant regions of the chimeric antibody are from a second species.

A “humanized antibody” as used herein refers to an antibody in which atleast one amino acid in a framework region of a non-human variableregion has been replaced with the corresponding amino acid from a humanvariable region. In some embodiments, a humanized antibody comprises atleast one human constant region or fragment thereof. In someembodiments, a humanized antibody is an Fab, an scFv, a (Fab′)₂, etc.

A “CDR-grafted antibody” as used herein refers to a humanized antibodyin which the complementarity determining regions (CDRs) of a first(non-human) species have been grafted onto the framework regions (FRs)of a second (human) species.

A “human antibody” as used herein refers to antibodies produced inhumans, antibodies produced in non-human animals that comprise humanimmunoglobulin genes, such as XenoMouse®, and antibodies selected usingin vitro methods, such as phage display, wherein the antibody repertoireis based on a human immunoglobulin sequences.

The term “leader sequence” refers to a sequence of amino acid residueslocated at the N terminus of a polypeptide that facilitates secretion ofa polypeptide from a mammalian cell. A leader sequence may be cleavedupon export of the polypeptide from the mammalian cell, forming a matureprotein. Leader sequences may be natural or synthetic, and they may beheterologous or homologous to the protein to which they are attached.Exemplary leader sequences include, but are not limited to, antibodyleader sequences, such as, for example, the amino acid sequences of SEQID NOs.: 3 and 4, which correspond to human light and heavy chain leadersequences, respectively. Nonlimiting exemplary leader sequences alsoinclude leader sequences from heterologous proteins. In someembodiments, an antibody lacks a leader sequence. In some embodiments,an antibody comprises at least one leader sequence, which may beselected from native antibody leader sequences and heterologous leadersequences.

The term “vector” is used to describe a polynucleotide that may beengineered to contain a cloned polynucleotide or polynucleotides thatmay be propagated in a host cell. A vector may include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that may be used in colorimetric assays,e.g., β-galactosidase). The term “expression vector” refers to a vectorthat is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6Eand DG44 cells, respectively.

The term “isolated” as used herein refers to a molecule that has beenseparated from at least some of the components with which it istypically found in nature. For example, a polypeptide is referred to as“isolated” when it is separated from at least some of the components ofthe cell in which it was produced. Where a polypeptide is secreted by acell after expression, physically separating the supernatant containingthe polypeptide from the cell that produced it is considered to be“isolating” the polypeptide. Similarly, a polynucleotide is referred toas “isolated” when it is not part of the larger polynucleotide (such as,for example, genomic DNA or mitochondrial DNA, in the case of a DNApolynucleotide) in which it is typically found in nature, or isseparated from at least some of the components of the cell in which itwas produced, e.g., in the case of an RNA polynucleotide. Thus, a DNApolynucleotide that is contained in a vector inside a host cell may bereferred to as “isolated” so long as that polynucleotide is not found inthat vector in nature.

The terms “subject” and “patient” are used interchangeably herein torefer to a human. In some embodiments, methods of treating othermammals, including, but not limited to, rodents, simians, felines,canines, equines, bovines, porcines, ovines, caprines, mammalianlaboratory animals, mammalian farm animals, mammalian sport animals, andmammalian pets, are also provided.

The term “rheumatoid arthritis” (“RA”) refers to a chronic autoimmunedisease characterized primarily by inflammation of the lining (synovium)of the joints, which can lead to joint damage, resulting in chronicpain, loss of function, and disability. Because RA can affect multipleorgans of the body, including skin, lungs, and eyes, it is referred toas a systemic illness.

The term “multiple sclerosis” (“MS”) refers to the chronic, autoimmune,demyelinating disease of the CNS in which the body generates antibodiesand white blood cells against the cells that produce the myelin sheath.“Demyelination” occurs when the myelin sheath becomes inflamed, injured,and detaches from the nerve fiber.

The term “cancer” refers to a proliferative disorder associated withuncontrolled cell proliferation, unrestrained cell growth, and decreasedcell deathiapoptosis. Cancer includes, but is not limited to, breastcancer, prostate cancer, lung cancer, kidney cancer, thyroid cancer,esophageal cancer, melanoma, follicular lymphomas, uveal melanoma, braincancer, head and neck cancer, pulmonary adenocarcinoma, including, butnot limited to, colon cancer, cardiac tumors, pancreatic cancer,retinoblastoma, glioblastoma, intestinal cancer, testicular cancer,stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,Kaposi's sarcoma, ovarian cancer, leukemia (including acute leukemias(for example, acute lymphocytic leukemia, acute myelocytic leukemia,including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (for example, chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia),myelodysplastic syndrome polycythemia vera, lymphomas (for example,Hodgkin's disease, non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain diseases, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer,endometrial cancer, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, and menangioma. The terms “metastasis” and“cancer metastasis” are used interchangeably herein to refer to theability of a cancer cell to spread to other tissues. For example,“metastasis to bone” refers to the ability of certain types of cancerincluding, but not limited to, breast, prostate, lung, kidney, thyroid,and melanoma, to metastasize to bone.

The term “osteolytic disorders” is used herein to refer to any conditionthat is caused by an increase in the activity of osteoclasts, which arecells responsible for bone resorption. The terms “osteolysis” and“osteolytic bone loss” may be used interchangeably to refer toosteoclast-mediated bone resorption or bone loss associated with anosteolytic disorder. Osteolytic disorders may occur in subjects with apredisposition to develop an osteolytic disorder, or they may occur insubjects with a disease that leads to or contributes to an osteolyticdisorder by stimulating osteoclast activity. In exemplary embodiments ofthe present invention, the osteolytic disorder may include osteolyticbone loss and cancer metastasis-induced osteolytic bone loss. In furtherexemplary embodiments of the present invention, the osteolytic bonedisorder includes metabolic bone disease, including endocrinopathies,such as hypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, and hyperthyroidism; dietary deficiency, includingrickets, osteomalacia, scurvy, and malnutrition; osteoporosis; drug use,including glucocorticoids (glucocorticoid-induced osteoporosis),heparin, and alcohol; chronic disease, including malabsorptionsyndromes; chronic renal failure, including renal osteodystrophy;chronic liver disease, including hepatic osteodystrophy; inheriteddisease, including osteogenesis imperfecta and homocystinuria; and boneinflammation associated with arthritis, rheumatoid arthritis, psoriaticarthritis, fibrous dysplasia, periodontal disease, and Paget's disease.

The terms “metastasis-induced osteolytic bone loss,” and “cancermetastasis-induced osteolytic bone loss,” are used interchangeablyherein to refer to osteolysis or osteolytic bone loss resulting fromcancer cell metastasis to bone. The term “cancer metastasis-inducedosteoclast activation” is used herein to refer to the ability of cancercells that have metastasized to bone to induce the activation ofosteoclasts.

The term “tumor” is used herein to refer to a group of cells thatexhibit abnormally high levels of proliferation and growth. A tumor maybe benign, pre-malignant, or malignant; malignant tumor cells arecancerous. Tumor cells may be solid tumor cells or leukemic tumor cells.The term “tumor growth” is used herein to refer to proliferation orgrowth by a cell or cells that comprise a tumor that leads to acorresponding increase in the size of the tumor. The term“CSF1R-dependent tumor growth” is used herein to refer to therequirement of a tumor cell or cells for CSF1R-mediated function(s) inorder for the tumor cell or cells to proliferate or grow.

“Treatment,” as used herein, covers any administration or application ofa therapeutic for disease in a mammal, including a human, and includesinhibiting the disease or progression of the disease, inhibiting orslowing the disease or its progression, arresting its development,partially or fully relieving the disease, or curing the disease, forexample, by causing regression, or restoring or repairing a lost,missing, or defective function; or stimulating an inefficient process.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed. For example, if the therapeutic agent is to be administeredorally, the carrier may be a gel capsule. If the therapeutic agent is tobe administered subcutaneously, the carrier ideally is not irritable tothe skin and does not cause injection site reaction.

Anti-CSF1R Antibodies

The present inventors have invented a new set of antibodies directedagainst CSF1R. Anti-CSF1R antibodies include, but are not limited to,humanized antibodies, chimeric antibodies, mouse antibodies, humanantibodies, and antibodies comprising the heavy chain and/or light chainCDRs discussed herein.

Exemplary Humanized Antibodies

In some embodiments, humanized antibodies that bind CSF1R are provided.Humanized antibodies are useful as therapeutic molecules becausehumanized antibodies reduce or eliminate the human immune response tonon-human antibodies (such as the human anti-mouse antibody (HAMA)response), which can result in an immune response to an antibodytherapeutic, and decreased effectiveness of the therapeutic.

Nonlimiting exemplary humanized antibodies include Ab1 through Ab16,described herein. Nonlimiting exemplary humanized antibodies alsoinclude antibodies comprising a heavy chain variable region of anantibody selected from Ab1 to Ab16 and/or a light chain variable regionof an antibody selected from Ab1 to Ab16. Nonlimiting exemplaryhumanized antibodies include antibodies comprising a heavy chainvariable region selected from SEQ ID NOs: 39 to 45 and/or a light chainvariable region selected from SEQ ID NOs: 46 to 52. Exemplary humanizedantibodies also include, but are not limited to, humanized antibodiescomprising heavy chain CDR1, CDR2, and CDR3, and/or light chain CDR1,CDR2, and CDR3 of an antibody selected from 0301, 0302, and 0311.

In some embodiments, a humanized anti-CSF1R antibody comprises heavychain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, and CDR3 ofan antibody selected from 0301, 0302, and 0311. Nonlimiting exemplaryhumanized anti-CSF1R antibodies include antibodies comprising sets ofheavy chain CDR1, CDR2, and CDR3 selected from: SEQ ID NOs: 15, 16, and17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27, 28, and 29.Nonlimiting exemplary humanized anti-CSF1R antibodies also includeantibodies comprising sets of light chain CDR1, CDR2, and CDR3 selectedfrom: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ IDNOs: 30, 31, and 32.

Nonlimiting exemplary humanized anti-CSF1R antibodies include antibodiescomprising the sets of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 in Table 1 (SEQ ID NOs shown; see Table 8 forsequences). Each row of Table 1 shows the heavy chain CDR1, CDR2, andCDR3, and light chain CDR1, CDR2, and CDR3 of an exemplary antibody.

TABLE 1 Heavy chain and light chain CDRs Heavy chain Light chain CDR1CDR2 CDR3 CDR1 CDR2 CDR3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 15 1617 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Further Exemplary Humanized Antibodies

In some embodiments, a humanized anti-CSF1R antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, and whereinthe antibody binds CSF1R. In some embodiments, a humanized anti-CSF1Rantibody comprises a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46to 52, wherein the antibody binds CSF1R. In some embodiments, ahumanized anti-CSF1R antibody comprises a heavy chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 9, 11, 13, and 39 to 45; and a light chain comprising avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52; wherein the antibody binds CSF1R.

As used herein, whether a particular polypeptide is, for example, atleast 95% identical to an amino acid sequence can be determined using,e.g., a computer program. When determining whether a particular sequenceis, for example, 95% identical to a reference sequence, the percentageof identity is calculated over the full length of the reference aminoacid sequence.

In some embodiments, a humanized anti-CSF1R antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, ahumanized anti-CSF1R antibody comprises at least one CDR selected from aheavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein,a heavy chain CDR3 discussed herein, a light chain CDR1 discussedherein, a light chain CDR2 discussed herein, and a light chain CDR3discussed herein. Further, in some embodiments, a humanized anti-CSF1Rantibody comprises at least one mutated CDR based on a CDR discussedherein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acidsubstitutions relative to the CDR discussed herein. In some embodiments,one or more of the amino acid substitutions are conservative amino acidsubstitutions. One skilled in the art can select one or more suitableconservative amino acid substitutions for a particular CDR sequence,wherein the suitable conservative amino acid substitutions are notpredicted to significantly alter the binding properties of the antibodycomprising the mutated CDR.

Exemplary humanized anti-CSF1R antibodies also include antibodies thatcompete for binding to CSF1R with an antibody described herein. Thus, insome embodiments, a humanized anti-CSF1R antibody is provided thatcompetes for binding to CSF1R with an antibody selected from Fabs 0301,0302, and 0311; and bivalent (i.e., having two heavy chains and twolight chains) antibody versions of those Fabs.

Exemplary Humanized Antibody Constant Regions

In some embodiments, a humanized antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a humanized antibodydescribed herein comprises a human IgG constant region. In someembodiments, a humanized antibody described herein comprises a humanIgG4 heavy chain constant region. In some such embodiments, a humanizedantibody described herein comprises an S241P mutation in the human IgG4constant region. In some embodiments, a humanized antibody describedherein comprises a human IgG4 constant region and a human κ light chain.

The choice of heavy chain constant region can determine whether or notan antibody will have effector function in vivo. Such effector function,in some embodiments, includes antibody-dependent cell-mediatedcytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), andcan result in killing of the cell to which the antibody is bound. Insome methods of treatment, including methods of treating some cancers,cell killing may be desirable, for example, when the antibody binds to acell that supports the maintenance or growth of the tumor. Exemplarycells that may support the maintenance or growth of a tumor include, butare not limited to, tumor cells themselves, cells that aid in therecruitment of vasculature to the tumor, and cells that provide ligands,growth factors, or counter-receptors that support or promote tumourgrowth or tumour survival. In some embodiments, when effector functionis desirable, an anti-CSF1R antibody comprising a human IgG1 heavy chainor a human IgG3 heavy chain is selected.

In some methods of treatment, effector function may not be desirable.For example, in some embodiments, it may be desirable that antibodiesused in the treatment of MS and/or RA and/or osteolysis do not haveeffector function. Thus, in some embodiments, anti-CSF1R antibodiesdeveloped for the treatment of cancer may not be suitable for use intreatment of MS and/or RA and/or osteolysis. Accordingly, in someembodiments, an anti-CSF1R antibody that lacks significant effectorfunction is used in treatment of MS and/or RA and/or osteolysis. In someembodiments, an anti-CSF1R antibody for treatment of MS and/or RA and/orosteolysis comprises a human IgG4 or IgG2 heavy chain constant region.In some embodiments, an IgG4 constant region comprises an S241Pmutation.

An antibody may be humanized by any method. Nonlimiting exemplarymethods of humanization include methods described, e.g., in U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; Jones etal., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-27(1988); Verhoeyen et al., Science 239: 1534-36 (1988); and U.S.Publication No. US 2009/0136500.

As noted above, a humanized antibody is an antibody in which at leastone amino acid in a framework region of a non-human variable region hasbeen replaced with the amino acid from the corresponding location in ahuman framework region. In some embodiments, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least 10, at least 11, at least 12, atleast 15, or at least 20 amino acids in the framework regions of anon-human variable region are replaced with an amino acid from one ormore corresponding locations in one or more human framework regions.

In some embodiments, some of the corresponding human amino acids usedfor substitution are from the framework regions of different humanimmunoglobulin genes. That is, in some such embodiments, one or more ofthe non-human amino acids may be replaced with corresponding amino acidsfrom a human framework region of a first human antibody or encoded by afirst human immunoglobulin gene, one or more of the non-human aminoacids may be replaced with corresponding amino acids from a humanframework region of a second human antibody or encoded by a second humanimmunoglobulin gene, one or more of the non-human amino acids may bereplaced with corresponding amino acids from a human framework region ofa third human antibody or encoded by a third human immunoglobulin gene,etc. Further, in some embodiments, all of the corresponding human aminoacids being used for substitution in a single framework region, forexample, FR2, need not be from the same human framework. In someembodiments, however, all of the corresponding human amino acids beingused for substitution are from the same human antibody or encoded by thesame human immunoglobulin gene.

In some embodiments, an antibody is humanized by replacing one or moreentire framework regions with corresponding human framework regions. Insome embodiments, a human framework region is selected that has thehighest level of homology to the non-human framework region beingreplaced. In some embodiments, such a humanized antibody is aCDR-grafted antibody.

In some embodiments, following CDR-grafting, one or more framework aminoacids are changed back to the corresponding amino acid in a mouseframework region. Such “back mutations” are made, in some embodiments,to retain one or more mouse framework amino acids that appear tocontribute to the structure of one or more of the CDRs and/or that maybe involved in antigen contacts and/or appear to be involved in theoverall structural integrity of the antibody. In some embodiments, tenor fewer, nine or fewer, eight or fewer, seven or fewer, six or fewer,five or fewer, four or fewer, three or fewer, two or fewer, one, or zeroback mutations are made to the framework regions of an antibodyfollowing CDR grafting.

In some embodiments, a humanized antibody also comprises a human heavychain constant region and/or a human light chain constant region.

Exemplary Chimeric Antibodies

In some embodiments, an anti-CSF1R antibody is a chimeric antibody. Insome embodiments, an anti-CSF1R antibody comprises at least onenon-human variable region and at least one human constant region. Insome such embodiments, all of the variable regions of an anti-CSF1Rantibody are non-human variable regions, and all of the constant regionsof an anti-CSF1R antibody are human constant regions. In someembodiments, one or more variable regions of a chimeric antibody aremouse variable regions. The human constant region of a chimeric antibodyneed not be of the same isotype as the non-human constant region, ifany, it replaces. Chimeric antibodies are discussed, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-55 (1984).

Nonlimiting exemplary chimeric antibodies include chimeric antibodiescomprising the heavy and/or light chain variable regions of an antibodyselected from 0301, 0302, and 0311. Additional nonlimiting exemplarychimeric antibodies include chimeric antibodies comprising heavy chainCDR1, CDR2, and CDR3, and/or light chain CDR1, CDR2, and CDR3 of anantibody selected from 0301, 0302, and 0311.

Nonlimiting exemplary chimeric anti-CSF1R antibodies include antibodiescomprising the following pairs of heavy and light chain variableregions: SEQ ID NOs: 9 and 10; SEQ ID NOs: 11 and 12; and SEQ ID NOs: 13and 14.

Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising a set of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 shown above in Table 1.

Further Exemplary Chimeric Antibodies

In some embodiments, a chimeric anti-CSF1R antibody comprises a heavychain comprising a variable region sequence that is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein theantibody binds CSF1R. In some embodiments, a chimeric anti-CSF1Rantibody comprises a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46to 52, wherein the antibody binds CSF1R. In some embodiments, a chimericanti-CSF1R antibody comprises a heavy chain comprising a variable regionsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to a sequence selected from SEQ ID NOs: 9, 11,13, and 39 to 45; and a light chain comprising a variable regionsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to a sequence selected from SEQ ID NOs: 10, 12,14, and 46 to 52; wherein the antibody binds CSF1R.

In some embodiments, a chimeric anti-CSF1R antibody comprises at leastone of the CDRs discussed herein. That is, in some embodiments, achimeric anti-CSF1R antibody comprises at least one CDR selected from aheavy chain CDR1 discussed herein, a heavy chain CDR2 discussed herein,a heavy chain CDR3 discussed herein, a light chain CDR1 discussedherein, a light chain CDR2 discussed herein, and a light chain CDR3discussed herein. Further, in some embodiments, a chimeric anti-CSF1Rantibody comprises at least one mutated CDR based on a CDR discussedherein, wherein the mutated CDR comprises 1, 2, 3, or 4 amino acidsubstitutions relative to the CDR discussed herein. In some embodiments,one or more of the amino acid substitutions are conservative amino acidsubstitutions. One skilled in the art can select one or more suitableconservative amino acid substitutions for a particular CDR sequence,wherein the suitable conservative amino acid substitutions are notpredicted to significantly alter the binding properties of the antibodycomprising the mutated CDR.

Exemplary chimeric anti-CSF1R antibodies also include chimericantibodies that compete for binding to CSF1R with an antibody describedherein. Thus, in some embodiments, a chimeric anti-CSF1R antibody isprovided that competes for binding to CSF1R with an antibody selectedfrom Fabs 0301, 0302, and 0311; and bivalent (i.e., having two heavychains and two light chains) antibody versions of those Fabs.

Exemplary Chimeric Antibody Constant Regions

In some embodiments, a chimeric antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG constant region. In someembodiments, a chimeric antibody described herein comprises a human IgG4heavy chain constant region. In some such embodiments, a chimericantibody described herein comprises an S241P mutation in the human IgG4constant region. In some embodiments, a chimeric antibody describedherein comprises a human IgG4 constant region and a human κ light chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, a chimericanti-CSF1R antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a chimericanti-CSF1R antibody comprising a human IgG4 or IgG2 heavy chain constantregion is selected.

Exemplary Human Antibodies

Human antibodies can be made by any suitable method. Nonlimitingexemplary methods include making human antibodies in transgenic micethat comprise human immunoglobulin loci. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993); Jakobovits et al., Nature362: 255-8 (1993); Lonberg et al., Nature 368: 856-9 (1994); and U.S.Pat. Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807;6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.

Nonlimiting exemplary methods also include making human antibodies usingphage display libraries. See, e.g., Hoogenboom et al., J Mol. Biol. 227:381-8 (1992); Marks et al., J. Mol. Biol. 222: 581-97 (1991); and PCTPublication No. WO 99/10494.

In some embodiments, a human anti-CSF1R antibody binds to a polypeptidehaving the sequence of SEQ ID NO: 1. Exemplary human anti-CSF1Rantibodies also include antibodies that compete for binding to CSF1Rwith an antibody described herein. Thus, in some embodiments, a humananti-CSF1R antibody is provided that competes for binding to CSF1R withan antibody selected from Fabs 0301, 0302, and 0311, and bivalent (i.e.,having two heavy chains and two light chains) antibody versions of thoseFabs.

In some embodiments, a human anti-CSF1R antibody comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, a human antibody describedherein comprises a human IgG constant region. In some embodiments, ahuman antibody described herein comprises a human IgG4 heavy chainconstant region. In some such embodiments, a human antibody describedherein comprises an S241P mutation in the human IgG4 constant region. Insome embodiments, a human antibody described herein comprises a humanIgG4 constant region and a human κ light chain.

In some embodiments, when effector function is desirable, a humananti-CSF1R antibody comprising a human IgG1 heavy chain constant regionor a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a human anti-CSF1Rantibody comprising a human IgG4 or IgG2 heavy chain constant region isselected.

Additional Exemplary Anti-CSF1R Antibodies

Exemplary anti-CSF1R antibodies also include, but are not limited to,mouse, humanized, human, chimeric, and engineered antibodies thatcomprise, for example, one or more of the CDR sequences describedherein. In some embodiments, an anti-CSF1R antibody comprises a heavychain variable region described herein. In some embodiments, ananti-CSF1R antibody comprises a light chain variable region describedherein. In some embodiments, an anti-CSF1R antibody comprises a heavychain variable region described herein and a light chain variable regiondescribed herein. In some embodiments, an anti-CSF1R antibody comprisesheavy chain CDR1, CDR2, and CDR3 described herein. In some embodiments,an anti-CSF1R antibody comprises light chain CDR1, CDR2, and CDR3described herein. In some embodiments, an anti-CSF1R antibody comprisesheavy chain CDR1, CDR2, and CDR3 described herein and light chain CDR1,CDR2, and CDR3 described herein.

In some embodiments, an anti-CSF1R antibody comprises a heavy chainvariable region of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising a heavy chain variable region of an antibody selected fromhumanized antibodies Ab1 to Ab16. Nonlimiting exemplary anti-CSF1Rantibodies include antibodies comprising a heavy chain variable regioncomprising a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

In some embodiments, an anti-CSF1R antibody comprises a light chainvariable region of an antibody selected from Fabs 0301, 0302, and 311.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising a light chain variable region of an antibody selected fromhumanized antibodies Ab1 to Ab16. Nonlimiting exemplary anti-CSF1Rantibodies include antibodies comprising a light chain variable regioncomprising a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52.

In some embodiments, an anti-CSF1R antibody comprises a heavy chainvariable region and a light chain variable region of an antibodyselected from Fabs 0301, 0302, and 0311. Nonlimiting exemplaryanti-CSF1R antibodies also include antibodies comprising a heavy chainvariable region and a light chain variable region of an antibodyselected from humanized antibodies Ab1 to Ab16. Nonlimiting exemplaryanti-CSF1R antibodies include antibodies comprising the following pairsof heavy and light chain variable regions: SEQ ID NOs: 9 and 10; SEQ IDNOs: 11 and 12; and SEQ ID NOs: 13 and 14; SEQ ID NOs: 39 and 40; SEQ IDNOs: 41 and 42; SEQ ID NOs: 43 and 44; SEQ ID NOs: 45 and 46; SEQ IDNOs: 47 and 48; SEQ ID NOs: 49 and 50; and SEQ ID NOs: 51 and 52.Nonlimiting exemplary anti-CSF1R antibodies also include antibodiescomprising the following pairs of heavy and light chains: SEQ ID NOs: 33and 34; SEQ ID NOs: 35 and 36; and SEQ ID NOs: 37 and 38.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1,CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising sets of heavy chain CDR1, CDR2, and CDR3 selected from: SEQID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22, and 23; and SEQ ID NOs: 27,28, and 29.

In some embodiments, an anti-CSF1R antibody comprises light chain CDR1,CDR2, and CDR3 of an antibody selected from Fabs 0301, 0302, and 0311.Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising sets of light chain CDR1, CDR2, and CDR3 selected from: SEQID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25, and 26; and SEQ ID NOs: 30,31, and 32.

In some embodiments, an anti-CSF1R antibody comprises heavy chain CDR1,CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 of an antibodyselected from Fabs 0301, 0302, and 0311.

Nonlimiting exemplary anti-CSF1R antibodies include antibodiescomprising the sets of heavy chain CDR1, CDR2, and CDR3, and light chainCDR1, CDR2, and CDR3 shown above in Table 1.

Further Exemplary Antibodies

In some embodiments, an anti-CSF1R antibody comprises a heavy chaincomprising a variable region sequence that is at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to a sequenceselected from SEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the antibodybinds CSF1R. In some embodiments, an anti-CSF1R antibody comprises alight chain comprising a variable region sequence that is at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to asequence selected from SEQ ID NOs: 10, 12, 14, and 46 to 52, wherein theantibody binds CSF1R. In some embodiments, an anti-CSF1R antibodycomprises a heavy chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to45; and a light chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to a sequence selected from SEQ ID NOs: 10, 12, 14, and 46 to52; wherein the antibody binds CSF1R.

In some embodiments, an anti-CSF1R antibody comprises at least one ofthe CDRs discussed herein. That is, in some embodiments, an anti-CSF1Rantibody comprises at least one CDR selected from a heavy chain CDR1discussed herein, a heavy chain CDR2 discussed herein, a heavy chainCDR3 discussed herein, a light chain CDR1 discussed herein, a lightchain CDR2 discussed herein, and a light chain CDR3 discussed herein.Further, in some embodiments, an anti-CSF1R antibody comprises at leastone mutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the antibody comprising the mutated CDR.

Exemplary anti-CSF1R antibodies also include antibodies that compete forbinding to CSF1R with an antibody described herein. Thus, in someembodiments, an anti-CSF1R antibody is provided that competes forbinding to CSF1R with an antibody selected from Fabs 0301, 0302, and0311, and bivalent (i.e., having two heavy chains and two light chains)antibody versions of those Fabs.

Exemplary Antibody Constant Regions

In some embodiments, an antibody described herein comprises one or morehuman constant regions. In some embodiments, the human heavy chainconstant region is of an isotype selected from IgA, IgG, and IgD. Insome embodiments, the human light chain constant region is of an isotypeselected from κ and λ. In some embodiments, an antibody described hereincomprises a human IgG constant region. In some embodiments, an antibodydescribed herein comprises a human IgG4 heavy chain constant region. Insome such embodiments, an antibody described herein comprises an S241Pmutation in the human IgG4 constant region. In some embodiments, anantibody described herein comprises a human IgG4 constant region and ahuman κ light chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, an anti-CSF1Rantibody comprising a human IgG1 heavy chain constant region or a humanIgG3 heavy chain constant region is selected. In some embodiments, wheneffector function is not desirable, an anti-CSF1R antibody comprising ahuman IgG4 or IgG2 heavy chain constant region is selected.

Exemplary Anti-CSF1R Heavy Chain Variable Regions

In some embodiments, anti-CSF1R antibody heavy chain variable regionsare provided. In some embodiments, an anti-CSF1R antibody heavy chainvariable region is a mouse variable region, a human variable region, ora humanized variable region.

An anti-CSF1R antibody heavy chain variable region comprises a heavychain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSF1Rantibody heavy chain variable region further comprises a heavy chain FR1and/or FR4. Nonlimiting exemplary heavy chain variable regions include,but are not limited to, heavy chain variable regions having an aminoacid sequence selected from SEQ ID NOs: 9, 11, 13, and 39 to 45.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR1 comprising a sequence selected from SEQ ID NOs: 15, 21,and 27.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR2 comprising a sequence selected from SEQ ID NOs: 16, 22,and 28.

In some embodiments, an anti-CSF1R antibody heavy chain variable regioncomprises a CDR3 comprising a sequence selected from SEQ ID NOs: 17, 23,and 29.

Nonlimiting exemplary heavy chain variable regions include, but are notlimited to, heavy chain variable regions comprising sets of CDR1, CDR2,and CDR3 selected from: SEQ ID NOs: 15, 16, and 17; SEQ ID NOs: 21, 22,and 23; and SEQ ID NOs: 27, 28, and 29.

In some embodiments, an anti-CSF1R antibody heavy chain comprises avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 9, 11, 13, and 39 to 45, wherein the heavy chain, togetherwith a light chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, an anti-CSF1R antibody heavy chain comprises atleast one of the CDRs discussed herein. That is, in some embodiments, ananti-CSF1R antibody heavy chain comprises at least one CDR selected froma heavy chain CDR1 discussed herein, a heavy chain CDR2 discussedherein, and a heavy chain CDR3 discussed herein. Further, in someembodiments, an anti-CSF1R antibody heavy chain comprises at least onemutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the heavy chain comprising the mutatedCDR.

In some embodiments, a heavy chain comprises a heavy chain constantregion. In some embodiments, a heavy chain comprises a human heavy chainconstant region. In some embodiments, the human heavy chain constantregion is of an isotype selected from IgA, IgG, and IgD. In someembodiments, the human heavy chain constant region is an IgG constantregion. In some embodiments, a heavy chain comprises a human igG4 heavychain constant region. In some such embodiments, the human IgG4 heavychain constant region comprises an S241P mutation.

In some embodiments, when effector function is desirable, a heavy chaincomprises a human IgG1 or IgG3 heavy chain constant region. In someembodiments, when effector function is less desirable, a heavy chaincomprises a human IgG4 or IgG2 heavy chain constant region.

Exemplary Anti-CSF1R Light Chain Variable Regions

In some embodiments, anti-CSF1R antibody light chain variable regionsare provided. In some embodiments, an anti-CSF1R antibody light chainvariable region is a mouse variable region, a human variable region, ora humanized variable region.

An anti-CSF1R antibody light chain variable region comprises a lightchain CDR1, FR2, CDR2, FR3, and CDR3. In some embodiments, an anti-CSF1Rantibody light chain variable region further comprises a light chain FR1and/or FR4. Nonlimiting exemplary light chain variable regions includelight chain variable regions having an amino acid sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR1 comprising a sequence selected from SEQ ID NOs: 18, 24and 30.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR2 comprising a sequence selected from SEQ ID NOs: 19, 25,and 31.

In some embodiments, an anti-CSF1R antibody light chain variable regioncomprises a CDR3 comprising a sequence selected from SEQ ID NOs: 20, 26,and 32.

Nonlimiting exemplary light chain variable regions include, but are notlimited to, light chain variable regions comprising sets of CDR1, CDR2,and CDR3 selected from: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 24, 25,and 26; and SEQ ID NOs: 30, 31, and 32.

In some embodiments, an anti-CSF1R antibody light chain comprises avariable region sequence that is at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence selected fromSEQ ID NOs: 10, 12, 14, and 46 to 52, wherein the light chain, togetherwith a heavy chain, is capable of forming an antibody that binds CSF1R.

In some embodiments, an anti-CSF1R antibody light chain comprises atleast one of the CDRs discussed herein. That is, in some embodiments, ananti-CSF1R antibody light chain comprises at least one CDR selected froma light chain CDR1 discussed herein, a light chain CDR2 discussedherein, and a light chain CDR3 discussed herein. Further, in someembodiments, an anti-CSF1R antibody light chain comprises at least onemutated CDR based on a CDR discussed herein, wherein the mutated CDRcomprises 1, 2, 3, or 4 amino acid substitutions relative to the CDRdiscussed herein. In some embodiments, one or more of the amino acidsubstitutions are conservative amino acid substitutions. One skilled inthe art can select one or more suitable conservative amino acidsubstitutions for a particular CDR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the light chain comprising the mutatedCDR.

In some embodiments, a light chain comprises a human light chainconstant region. In some embodiments, a human light chain constantregion is selected from a human κ and a human λ light chain constantregion.

Exemplary Additional CSF1R Binding Molecules

In some embodiments, additional molecules that bind CSF1R are provided.Such molecules include, but are not limited to, non-canonical scaffolds,such as anti-calins, adnectins, ankyrin repeats, etc. See, e.g., Hosseet al., Prot. Sci. 15:14 (2006); Fiedler, M. and Skerra, A.,“Non-Antibody Scaffolds,” pp. 467-499 in Handbook of TherapeuticAntibodies, Dubel, S., ed., Wiley-VCH, Weinheim, Germany, 2007.

Exemplary Properties of Anti-CSF1R Antibodies

In some embodiments, an antibody having a structure described abovebinds to the CSF1R with a binding affinity (K_(D)) of less than 1 nM,blocks binding of CSF1 and/or IL34 to CSF1R, and inhibits CSF1Rphosphorylation induced by CSF1 and/or IL34.

In some embodiments, an anti-CSF1R antibody binds to the extracellulardomain of CSF1R (CSF1R-ECD). In some embodiments, an anti-CSF1R antibodyhas a binding affinity (K_(D)) for CSF1R of less than 1 nM, less than0.5 nM, less than 0.1 nM, or less than 0.05 nM. In some embodiments, ananti-CSF1R antibody has a K_(D) of between 0.01 and 1 nM, between 0.01and 0.5 nM, between 0.01 and 0.1 nM, between 0.01 and 0.05 nM, orbetween 0.02 and 0.05 nM.

In some embodiments, an anti-CSF1R antibody blocks ligand binding toCSF1R. In some embodiments, an anti-CSF1R antibody blocks binding ofCSF1 to CSF1R. In some embodiments, an anti-CSF1R antibody blocksbinding of IL34 to CSF1R. In some embodiments, an anti-CSF1R antibodyblocks binding of both CSF1 and IL34 to CSF1R. In some embodiments, anantibody that blocks ligand binding binds to the extracellular domain ofCSF1R. An antibody is considered to “block ligand binding to CSF1R” whenit reduces the amount of detectable binding of a ligand to CSF1R by atleast 50%, using the assay described in Example 7. In some embodiments,an antibody reduces the amount of detectable binding of a ligand toCSF1R by at least 60%, at least 70%, at least 80%, or at least 90%,using the assay described in Example 7. In some such embodiments, theantibody is said to block ligand binding by at least 50%, at least 60%,at least 70%, etc.

In some embodiments, an anti-CSF1R antibody inhibits ligand-inducedCSF1R phosphorylation. In some embodiments, an anti-CSF1R antibodyinhibits CSF1-induced CSF1R phosphorylation. In some embodiments, ananti-CSF1R antibody inhibits IL34-induced CSF1R phosphorylation. In someembodiments, an anti-CSF1R antibody inhibits both CSF1-induced andIL34-induced CSF1R phosphorylation. An antibody is considered to“inhibit ligand-induced CSF1R phosphorylation” when it reduces theamount of detectable ligand-induced CSF1R phosphorylation by at least50%, using the assay described in Example 6. In some embodiments, anantibody reduces the amount of detectable ligand-induced CSF1Rphosphorylation by at least 60%, at least 70%, at least 80%, or at least90%, using the assay described in Example 6. In some such embodiments,the antibody is said to inhibit ligand-induced CSF1R phosphorylation byat least at least 50%, at least 60%, at least 70%, etc.

In some embodiments, an antibody inhibits monocyte proliferation and/orsurvival responses in the presence of CSF1 and/or IL34. An antibody isconsidered to “inhibit monocyte proliferation and/or survival responses”when it reduces the amount of monocyte proliferation and/or survivalresponses in the presence of CSF1 and/or IL34 by at least 50%, using theassay described in Example 10. In some embodiments, an antibody reducesthe amount of monocyte proliferation and/or survival responses in thepresence of CSF1 and/or IL34 by at least 60%, at least 70%, at least80%, or at least 90%, using the assay described in Example 10. In somesuch embodiments, the antibody is said to inhibit monocyte proliferationand/or survival responses by at least at least 50%, at least 60%, atleast 70%, etc.

Exemplary Antibody Conjugates

In some embodiments, an anti-CSF1R antibody is conjugated to a labeland/or a cytotoxic agent. As used herein, a label is a moiety thatfacilitates detection of the antibody and/or facilitates detection of amolecule to which the antibody binds. Nonlimiting exemplary labelsinclude, but are not limited to, radioisotopes, fluorescent groups,enzymatic groups, chemiluminescent groups, biotin, epitope tags,metal-binding tags, etc. One skilled in the art can select a suitablelabel according to the intended application.

As used herein, a cytotoxic agent is a moiety that reduces theproliferative capacity of one or more cells. A cell has reducedproliferative capacity when the cell becomes less able to proliferate,for example, because the cell undergoes apoptosis or otherwise dies, thecell fails to proceed through the cell cycle and/or fails to divide, thecell differentiates, etc. Nonlimiting exemplary cytotoxic agentsinclude, but are not limited to, radioisotopes, toxins, andchemotherapeutic agents. One skilled in the art can select a suitablecytotoxic according to the intended application.

In some embodiments, a label and/or a cytotoxic agent is conjugated toan antibody using chemical methods in vitro. Nonlimiting exemplarychemical methods of conjugation are known in the art, and includeservices, methods and/or reagents commercially available from, e.g.,Thermo Scientific Life Science Research Produces (formerly Pierce;Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI Antibody Services(Calgary, Canada), AbD Serotec (Raleigh, N.C.), etc. In someembodiments, when a label and/or cytotoxic agent is a polypeptide, thelabel and/or cytotoxic agent can be expressed from the same expressionvector with at least one antibody chain to produce a polypeptidecomprising the label and/or cytotoxic agent fused to an antibody chain.One skilled in the art can select a suitable method for conjugating alabel and/or cytotoxic agent to an antibody according to the intendedapplication.

Exemplary Leader Sequences

In order for some secreted proteins to express and secrete in largequantities, a leader sequence from a heterologous protein may bedesirable. In some embodiments, a leader sequence is selected from SEQID NOs: 3 and 4, which are light chain and heavy chain leader sequences,respectively. In some embodiments, employing heterologous leadersequences may be advantageous in that a resulting mature polypeptide mayremain unaltered as the leader sequence is removed in the ER during thesecretion process. The addition of a heterologous leader sequence may berequired to express and secrete some proteins.

Certain exemplary leader sequence sequences are described, e.g., in theonline Leader sequence Database maintained by the Department ofBiochemistry, National University of Singapore. See Choo et al., BMCBioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.

Nucleic Acid Molecules Encoding Anti-CSF1R Antibodies

Nucleic acid molecules comprising polynucleotides that encode one ormore chains of anti-CSF1R antibodies are provided. In some embodiments,a nucleic acid molecule comprises a polynucleotide that encodes a heavychain or a light chain of an anti-CSF1R antibody. In some embodiments, anucleic acid molecule comprises both a polynucleotide that encodes aheavy chain and a polynucleotide that encodes a light chain, of ananti-CSF1R antibody. In some embodiments, a first nucleic acid moleculecomprises a first polynucleotide that encodes a heavy chain and a secondnucleic acid molecule comprises a second polynucleotide that encodes alight chain.

In some such embodiments, the heavy chain and the light chain areexpressed from one nucleic acid molecule, or from two separate nucleicacid molecules, as two separate polypeptides. In some embodiments, suchas when an antibody is an scFv, a single polynucleotide encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether.

In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-CSF1R antibody comprises a nucleotide sequence thatencodes a leader sequence, which, when translated, is located at the Nterminus of the heavy chain or light chain. As discussed above, theleader sequence may be the native heavy or light chain leader sequence,or may be another heterologous leader sequence.

Nucleic acid molecules may be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Anti-CSF1R Antibody Expression and Production

Vectors

Vectors comprising polynucleotides that encode anti-CSF1R heavy chainsand/or anti-CSF1R light chains are provided. Vectors comprisingpolynucleotides that encode anti-CSF1R heavy chains and/or anti-CSF1Rlight chains are also provided. Such vectors include, but are notlimited to, DNA vectors, phage vectors, viral vectors, retroviralvectors, etc. In some embodiments, a vector comprises a firstpolynucleotide sequence encoding a heavy chain and a secondpolynucleotide sequence encoding a light chain. In some embodiments, theheavy chain and light chain are expressed from the vector as twoseparate polypeptides. In some embodiments, the heavy chain and lightchain are expressed as part of a single polypeptide, such as, forexample, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, e.g., in Running Deer et al.,Biotechnol. Prog. 20:880-889 (2004).

In some embodiments, a vector is chosen for in vivo expression ofanti-CSF1R heavy chains and/or anti-CSF1R light chains in animals,including humans. In some such embodiments, expression of thepolypeptide is under the control of a promoter that functions in atissue-specific manner. For example, liver-specific promoters aredescribed, e.g., in PCT Publication No. WO 2006/076288.

Host Cells

In various embodiments, anti-CSF1R heavy chains and/or anti-CSF1R lightchains may be expressed in prokaryotic cells, such as bacterial cells;or in eukaryotic cells, such as fungal cells (such as yeast), plantcells, insect cells, and mammalian cells. Such expression may be carriedout, for example, according to procedures known in the art. Exemplaryeukaryotic cells that may be used to express polypeptides include, butare not limited to, COS cells, including COS 7 cells; 293 cells,including 293-6E cells; CHO cells, including CHO-S and DG44 cells;PER.C6® cells (Crucell); and NSO cells. In some embodiments, anti-CSF1Rheavy chains and/or anti-CSF1R light chains may be expressed in yeast.See, e.g., U.S. Publication No. US 2006/0270045 A1. In some embodiments,a particular eukaryotic host cell is selected based on its ability tomake desired post-translational modifications to the anti-CSF1R heavychains and/or anti-CSF1R light chains. For example, in some embodiments,CHO cells produce polypeptides that have a higher level of sialylationthan the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. Cold SpringHarbor Laboratory Press (2001). Nucleic acids may be transiently orstably transfected in the desired host cells, according to any suitablemethod.

In some embodiments, one or more polypeptides may be produced in vivo inan animal that has been engineered or transfected with one or morenucleic acid molecules encoding the polypeptides, according to anysuitable method.

Purification of Anti-CSF1R Antibodies

Anti-CSF1R antibodies may be purified by any suitable method. Suchmethods include, but are not limited to, the use of affinity matrices orhydrophobic interaction chromatography. Suitable affinity ligandsinclude the CSF1R ECD and ligands that bind antibody constant regions.For example, a Protein A, Protein G, Protein A/G, or an antibodyaffinity column may be used to bind the constant region and to purify ananti-CSF1R antibody. Hydrophobic interactive chromatography, forexample, a butyl or phenyl column, may also suitable for purifying somepolypeptides. Many methods of purifying polypeptides are known in theart. Cell-free Production of Anti-CSF1R Antibodies

In some embodiments, an anti-CSF1R antibody is produced in a cell-freesystem. Nonlimiting exemplary cell-free systems are described, e.g., inSitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, TrendsBiotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21: 695-713(2003).

Therapeutic Compositions and Methods

Methods of Treating Diseases using Anti-CSF1R Antibodies

Antibodies of the invention, and compositions comprising antibodies ofthe invention, are provided for use in methods of treatment for humansor animals. Methods of treating disease comprising administeringanti-CSR1R antibodies are also provided. Nonlimiting exemplary diseasesthat can be treated with anti-CSF1R antibodies include, but are notlimited to, RA, MS, cancer, metastasis-induced osteolytic bone loss,osteolytic disorders, and hypercalcemia-induced bone loss.

In some embodiments, methods of treating inflammatory conditionscomprising administering an anti-CSF1R antibody are provided. In someembodiments, an inflammatory condition is selected from psoriasis, SLE(lupus), COPD, atopic dermatitis, and atherosclerosis, macrophageactivation syndrome, and histiocytosis X.

In some embodiments, methods of treating an inflammatory conditioncomprising administering an anti-CSF1R antibody are provided, whereinthe inflammatory condition is selected from: proliferative vasculardisease, acute respiratory distress syndrome, cytokine-mediatedtoxicity, interleukin-2 toxicity, appendicitis, peptic, gastric andduodenal ulcers, peritonitis, pancreatitis, ulcerative,pseudomembranous, acute and ischemic colitis, diverticulitis,epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis,inflammatory bowel disease, Crohn's disease, enteritis, Whipple'sdisease, asthma, allergy, anaphylactic shock, immune complex disease,organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis,septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilicgranuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis,vaginitis, prostatitis, urethritis, bronchitis, emphysema, rhinitis,cystic fibrosis, pneumonitis, alvealitis, bronchiolitis, pharyngitis,pleurisy, sinusitis, influenza, respiratory syncytial virus infection,herpes infection, HIV infection, hepatitis B virus infection, hepatitisC virus infection, disseminated bacteremia, Dengue fever, candidiasis,malaria, filariasis, amebiasis, hydatid cysts, burns, dermatitis,dermatomyositis, sunburn, urticaria, warts, wheals, vasulitis, angiitis,endocarditis, arteritis, atherosclerosis, thrombophlebitis,pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa,rheumatic fever, Alzheimer's disease, celiac disease, congestive heartfailure, meningitis, encephalitis, cerebral infarction, cerebralembolism, Guillain-Barre syndrome, neuritis, neuralgia, spinal cordinjury, paralysis, uveitis, arthritides, arthralgias, osteomyelitis,fasciitis, Paget's disease, gout, periodontal disease, synovitis,myasthenia gravis, thryoiditis, systemic lupus erythematosus,Goodpasture's syndrome, Behcets's syndrome, allograft rejection,graft-versus-host disease, ankylosing spondylitis, Berger's disease,type I diabetes, type 2 diabetes, Berger's disease, Retier's syndrome,and Hodgkins disease, or in treating inflammation associated with theseconditions.

In some embodiments, methods of treating cancer comprising administeringan anti-CSF1R antibody are provided. In some embodiments, the cancer isa CSF1-secreting cancer. In some embodiments, the cancer is one or morecancers selected from breast cancer, prostate cancer, endometrialcancer, bladder cancer, kidney cancer, esophageal cancer, squamous cellcarcinoma, uveal melanoma, follicular lymphoma, renal cell carcinoma,cervical cancer, and ovarian cancer. In some embodiments, an anti-CSF1Rantibody is useful for treating one or more cancers selected from lungcancer, colorectal cancer, brain cancer, pancreatic cancer, head andneck cancer, liver cancer, leukemia, lymphoma, Hodgkin's disease,multiple myeloma, melanoma, astrocytoma, stomach cancer, and pulmonaryadenocarcinoma.

Routes of Administration and Carriers

In various embodiments, anti-CSF1R antibodies may be administered invivo by various routes, including, but not limited to, oral,intra-arterial, parenteral, intranasal, intramuscular, intracardiac,intraventricular, intratracheal, buccal, rectal, intraperitoneal,intradermal, topical, transdermal, and intrathecal, or otherwise byimplantation or inhalation. The subject compositions may be formulatedinto preparations in solid, semi-solid, liquid, or gaseous forms;including, but not limited to, tablets, capsules, powders, granules,ointments, solutions, suppositories, enemas, injections, inhalants, andaerosols. A nucleic acid molecule encoding an anti-CSF1R antibody may becoated onto gold microparticles and delivered intradermally by aparticle bombardment device, or “gene gun,” as described in theliterature (see, e.g., Tang et al., Nature 356:152-154 (1992)). Theappropriate formulation and route of administration may be selectedaccording to the intended application.

In various embodiments, compositions comprising anti-CSF1R antibodiesare provided in formulations with a wide variety of pharmaceuticallyacceptable carriers (see, e.g., Gennaro, Remington: The Science andPractice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20thed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) ed., Lippencott Williams and Wilkins (2004); Kibbe etal., Handbook of Pharmaceutical Excipients, 3^(rd) ed., PharmaceuticalPress (2000)). Various pharmaceutically acceptable carriers, whichinclude vehicles, adjuvants, and diluents, are available. Moreover,various pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,wetting agents and the like, are also available. Non-limiting exemplarycarriers include saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof.

In various embodiments, compositions comprising anti-CSF1R antibodiesmay be formulated for injection, including subcutaneous administration,by dissolving, suspending, or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids, or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. In various embodiments, the compositions may beformulated for inhalation, for example, using pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen, and thelike. The compositions may also be formulated, in various embodiments,into sustained release microcapsules, such as with biodegradable ornon-biodegradable polymers. A non-limiting exemplary biodegradableformulation includes poly lactic acid-glycolic acid polymer. Anon-limiting exemplary non-biodegradable formulation includes apolyglycerin fatty acid ester. Certain methods of making suchformulations are described, for example, in EP 1 125 584 A1.

Pharmaceutical packs and kits comprising one or more containers, eachcontaining one or more doses of an anti-CSF1R antibody are alsoprovided. In some embodiments, a unit dosage is provided wherein theunit dosage contains a predetermined amount of a composition comprisingan anti-CSF1R antibody, with or without one or more additional agents.In some embodiments, such a unit dosage is supplied in single-useprefilled syringe for injection. In various embodiments, the compositioncontained in the unit dosage may comprise saline, sucrose, or the like;a buffer, such as phosphate, or the like; and/or be formulated within astable and effective pH range. Alternatively, in some embodiments, thecomposition may be provided as a lyophilized powder that may bereconstituted upon addition of an appropriate liquid, for example,sterile water. In some embodiments, the composition comprises one ormore substances that inhibit protein aggregation, including, but notlimited to, sucrose and arginine. In some embodiments, a composition ofthe invention comprises heparin and/or a proteoglycan.

Pharmaceutical compositions are administered in an amount effective fortreatment or prophylaxis of the specific indication. The therapeuticallyeffective amount is typically dependent on the weight of the subjectbeing treated, his or her physical or health condition, theextensiveness of the condition to be treated, or the age of the subjectbeing treated. In general, anti-CSF1R antibodies may be administered inan amount in the range of about 10 μg/kg body weight to about 100 mg/kgbody weight per dose. In some embodiments, anti-CSF1R antibodies may beadministered in an amount in the range of about 50 μg/kg body weight toabout 5 mg/kg body weight per dose. In some embodiments, anti-CSF1Rantibodies may be administered in an amount in the range of about 100μg/kg body weight to about 10 mg/kg body weight per dose. In someembodiments, anti-CSF1R antibodies may be administered in an amount inthe range of about 100 μg/kg body weight to about 20 mg/kg body weightper dose. In some embodiments, anti-CSF1R antibodies may be administeredin an amount in the range of about 0.5 mg/kg body weight to about 20mg/kg body weight per dose.

The anti-CSF1R antibody compositions may be administered as needed tosubjects. Determination of the frequency of administration may be madeby persons skilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like. In some embodiments,an effective dose of an anti-CSF1R antibody is administered to a subjectone or more times. In various embodiments, an effective dose of ananti-CSF1R antibody is administered to the subject once a month, morethan once a month, such as, for example, every two months or every threemonths. In other embodiments, an effective dose of an anti-CSF1Rantibody is administered less than once a month, such as, for example,every two weeks or every week. An effective dose of an anti-CSF1Rantibody is administered to the subject at least once. In someembodiments, the effective dose of an anti-CSF1R antibody may beadministered multiple times, including for periods of at least a month,at least six months, or at least a year.

Combination Therapy

Anti-CSF1R antibodies may be administered alone or with other modes oftreatment. They may be provided before, substantially contemporaneouswith, or after other modes of treatment, for example, surgery,chemotherapy, radiation therapy, or the administration of a biologic,such as another therapeutic antibody. For treatment of rheumatoidarthritis, anti-CSF1R antibodies may be administered with othertherapeutic agents, for example, methotrexate, anti-TNF agents such asRemicade, Humira, Simponi, and Enbrel; glucocorticoids such asprednisone; Leflunomide; Azothioprine; JAK inhibitors such as CP 590690;SYK inhibitors such as R788; anti-IL-6 antibodies; anti-IL-6Rantibodies; anti-CD-20 antibodies; anti-CD19 antibodies; anti-GM-CSFantibodies; and anti-GM-CSF-R antibodies. For treatment of multiplesclerosis, anti-CSF1R antibodies may be administered with othertherapeutic agents, for example, interferon alpha; interferon beta;prednisone; anti-alpha4 integrin antibodies such as Tysabri; anti-CD20antibodies such as Rituxan; FTY720 (Fingolimod); and Cladribine(Leustatin).

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1: Selection of Fabs that Bind CSF1R Extracellular Domain (ECD)

Mice were immunized with a human CSF1R extracellular domain Fc fusion,hCSF1R ECD.506-Fc (SEQ ID NO: 6). Spleens from immunized mice wereisolated, and a Fab phage display library was created from thespenocytes. Fab-expressing phage were selected for binding to humanCSF1R ECD. Fabs from positive-binding phage were expressed and purifiedfrom bacteria. A total of 1056 Fab clones were selected for furtheranalysis.

Fabs were screened for the ability to bind human CSF1R ECD, blockbinding of human CSF1 to human CSF1R ECD, and block binding of humanIL34 to human CSF1R ECD. Sequence analysis and clustering of the Fabsthat were selected from that screen was then performed and certainunique Fabs were selected.

The unique Fabs were further analyzed for the ability to bind humanCSF1R ECD, the ability to bind cynomolgus CSF1R ECD, and the ability tobind mouse CSF1R ECD. The Fabs were also analyzed for the ability toblock human CSF1 binding to human CSF1R ECD and the ability to blockhuman IL34 binding to human CSF1R ECD, and the ability to inhibitligand-induced CSF1R phosphorylation in the presence of CSF1 or IL34.(Data not shown.)

Example 2: Reformatting of Anti-CSF1R Fabs to Make Chimeric Antibodies

Following the Fab characterization, eleven of the Fabs were selected forreformatting to chimeric antibodies. Each Fab was reformatted to achimeric antibody comprising a human IgG4 heavy chain constant regionwith the S241P mutation, and a human κ light chain constant region.Briefly, Fab VH regions cloned into and expressed from vector pTT5(Biotechnology Research Institute, Montreal, Canada; and NationalResearch Council of Canada, Ottawa, Canada) modified to contain a mouseIgH leader sequence (SEQ ID NO: 4) and a human IgG4 heavy chain constantregion with the S241P mutation (SEQ ID NO: 94). Fab VL regions werecloned into and expressed from vector pTT5 modified to contain a mouseIgκ leader sequence (SEQ ID NO: 3) and a human Igκ light chain constantregion (SEQ ID NO: 95). Fab V regions were inserted in such a way as notto introduce non-antibody derived amino acid sequences into the finalproteins.

Example 3: Expression and Characterization of Chimeric Antibodies

The chimeric antibodies were transiently expressed and purifiedsubstantially as described below in Example 5.

The 11 chimeric antibodies were assayed for binding to human,cynomolgus, and mouse CSF1R ECD. The chimeric antibodies were alsoassayed for the ability to block binding of human CSF1 to human CSF1RECD, the ability to block binding of human IL34 to human CSF1R ECD, theability to block binding of human CSF1 to cynomolgus CSF1R ECD, and theability to inhibit ligand-induced CSF1R phosphorylation in the presenceof CSF1 or IL34. The chimeric antibodies were further assayed forbinding to CSF1R on the surface of cells. Finally, the chimericantibodies were assayed to confirm that they do not induce CSF1Rphosphorylation in the absence of ligand. (Data not shown.)

Example 4: Humanization of Anti-CSF1R Antibodies

From the analyses described above, chimeric anti-CSF1R antibodies 0301,0302, and 0311 were selected for humanization. The antibodies werehumanized by changing certain amino acid residues in the frameworkregions of the heavy and light chain variable regions. The criteria usedfor humanization were as described previously, e.g., in U.S. PublicationNo. US 2009/0136500.

For cAb 0301, three humanized heavy chain variable regions and twohumanized light chain variable regions were designed, for a total of sixhumanized antibodies, Ab1 to Ab6. For cAb 0302, two humanized heavychain variable regions and three humanized light chain variable regionswere designed, for a total of six humanized antibodies, Ab7 to Ab12. ForcAb 0311, two humanized heavy chain variable regions and two humanizedlight chain variable regions were designed, for a total of fourhumanized antibodies, Ab13 to Ab16.

The sequences for each of the humanized heavy chain variable regions andhumanized light chain variable regions, aligned with the sequences ofthe parental chimeric antibody variable regions and the sequences of thehuman acceptor variable framework regions are shown in FIGS. 1 (heavychains) and 2 (light chains). The changes in humanized variable regionsequences relative to the human acceptor variable framework regionsequences are boxed. Each of the CDRs for each of the variable regionsis shown in a boxed region, and labeled as “CDR” above the boxedsequences.

Table 8, below, shows the full sequences for the humanized heavy chainsand humanized light chains of antibodies Ab1 to Ab16. The name and SEQID NOs of the humanized heavy chain and humanized light chain of each ofthose antibodies is shown in Table 2.

TABLE 2 Humanized heavy chains and light chains of Ab1 to Ab16 HumanizedHumanized SEQ ID Humanized SEQ ID antibody HC NO LC NO Abl h0301-H0 53h0301-L0 60 Ab2 h0301-H1 54 h0301-L0 60 Ab3 h0301-H2 55 h0301-L0 60 Ab4h0301-H0 53 h0301-L1 61 Ab5 h0301-H1 54 h0301-L1 61 Ab6 h0301-H2 55h0301-L1 61 Ab7 h0302-H1 56 h0302-L0 62 Ab8 h0302-H1 56 h0302-L1 63 Ab9h0302-H1 56 h0302-L2 64 Ab10 h0302-H2 57 h0302-L0 62 Ab11 h0302-H2 57h0302-L1 63 Ab12 h0302-H2 57 h0302-L2 64 Ab13 h0311-H1 58 h0311-L0 65Ab14 h0311-H1 58 h0311-L1 66 Ab15 h0311-H2 59 h0311-L0 65 Ab16 h0311-H259 h0311-L1 66

Example 5: Humanized Anti-CSF1R Antibodies Bind to Human and CynomolgusCSF1R ECD, but not to Mouse CSF1R ECD

The 16 humanized antibodies were transiently expressed in CHO cells, asfollows. CHO-3E7 cells were co-transfected with individual heavy andlight chain expression plasmids at a mass ratio of 1 heavy chain plasmidto 2 light chain plasmids using polyethyleneinimine (PEI) at a DNA:PEIratio of 1:5. Total DNA used per transfection was 1.5 μg/ml of cells.

Humanized antibodies were purified from transfected cell supernatantsusing HiTrap Protein A HP columns (GE Healthcare) followed by furtherpurification using Phenyl HP columns (GE Healthcare). Antibodycontaining supernatants were loaded onto HiTrap Protein A HP columnspre-equilibrated with PBS/0.5M NaCl. Antibody loaded columns were washedwith 10 column volumes PBS/0.5M NaCl, and eluted with a mixedlinear-step gradient of 0.1 M Glycine, pH 2.7/0.5 M NaCl directly into100 ul of 1M Tris buffer, pH 8.0. Antibody containing eluates weredialyzed against PBS, after which 2.4 M (NH₄)₂SO₄ (Sigma) was added toachieve a conductivity equal to that of 10 mM Potassium PhosphatepH7.0/1.2 M (NH₄)₂SO₄. Antibodies were then loaded on 1 ml Phenyl HPcolumns (GE Healthcare) pre-equilibrated with 10 mM Potassium PhosphatepH7.0/1.2 M (NH₄)₂SO₄. Antibody loaded columns were washed with 15column volumes 10 mM Potassium Phosphate pH7.0/1.2 M (NH₄)₂SO₄, andeluted with a 20 column volume gradient of 10 mM Potassium Phosphate, pH7.0. Antibody containing fractions were pooled and dialyzed against PBS.

The humanized antibodies, along with their parental chimeric antibodies(cAbs), were assayed for binding to human, cynomolgus, and mouse CSF1RECD, as follows.

Human CSF1R Binding Activity

Ninety-six well clear-bottom ELISA plates were coated overnight with 1μg/ml recombinant hCSF1R ECD.506-Fc (SEQ ID NO: 6; FivePrimeTherapeutics) or Human M-CSF R Fc Chimera (R&D Systems) in PBS. The nextmorning, wells were washed four times with 0.05% Tween20 in PBS (PBST)and blocked with Blocker-Blotto (Pierce). Fifty μl of 0.5× serialdilutions of the humanized antibody or parental chimeric antibody,beginning with 2000 ng/ml, diluted 1:1 in Blocker-Blotto were added tothe CSF1R-coated wells. After incubation at room temperature (RT) for 90min, wells were washed four times with PBST, and a 1:5000 dilution of aperoxidase-conjugated Goat anti-Human kappa Light chain antibody (Sigma)in Blocker-Blotto was added to each well. After incubation at RT for 60min, wells were washed four times with PBST, and 50 μlo-phenylenediamine dihydrochloride peroxidase substrate (Sigma) wasadded to each well. After incubation at RT for 30 min, A450 values ofeach well were read directly on a SpectraMaxPlus spectrophotometer withSoftMaxPro software (Molecular Devices).

The results of that experiment is shown in FIG. 3. All of the humanizedantibodies bound to human CSF1R ECD within the range of concentrationstested.

Cynomolgus CSF1R Binding Curve

The binding curve for each humanized antibody binding to cynomolgusCSF1R ECD was determined as described above for human CSF1R, except thewells of the clear-bottom ELISA plates were coated overnight with 2μg/ml recombinant cynoCSF1R ECD-Fc (FivePrime Therapeutics, SEQ ID NO:8, but without the 19 amino acid leader sequence).

The results of that experiment are shown in FIG. 4. All of the humanizedantibodies bound to cynomolgus CSF1R ECD within the range ofconcentrations tested.

Mouse CSF1R Binding Curve

The binding curve for each humanized antibody binding to mouse CSF1R ECDwas determined as described above for human CSF1R, except the wells ofthe clear-bottom ELISA plates were coated overnight with 2 μg/mlrecombinant mCSF1R ECD-Fc (FivePrime Therapeutics, SEQ ID NO: 93).

The results of that experiment are shown in FIG. 5. None of thehumanized antibodies, or the parental chimeric antibodies, detectablybound to mouse CSF1R ECD over the range of concentrations tested.

Calculation of EC50s

Table 3 shows the EC50, calculated using the non-linear regression(curve-fit) analysis algorithm of the GraphPad Prism software (GraphPadSoftware) for each humanized antibody binding to human CSF1R ECD andcynomolgus CSF1R ECD. Because none of the chimeric antibodies detectablybound to mouse CSF1R ECD, an EC50 could not be calculated from thatdata. Table 3 also includes the calculated EC50s for the parentalchimeric antibodies.

TABLE 3 Binding activity of humanized anti-CSF1R antibodies HumanCynomolgus CSF1R CSF1R Humanized ECD EC50 ECD EC50 antibody (ng/ml)(ng/ml) cAb 0301 11.4 15.18 h0301-L0H0 13.4 15.11 h0301-L0H1 14.23 14.39h0301-L0H2 14.77 13.79 h0301-L1H0 13.35 11.93 h0301-L1H1 16.47 16.66h0301-L1H2 16.23 16.59 cAb 0302 15.94 17.34 h0302-L0H1 14.64 466.5h0302-L1H1 21.43 1058 h0302-L2H1 7.741 66.04 h0302-L0H2 17.85 154.9h0302-L1H2 22.1 172.5 h0302-L2H2 10.15 17.96 cAb 0311 17.65 20.06h0311-L0H1 13.12 21.65 h0311-L1H1 14.32 30.88 h0311-L0H2 11.54 17.47h0311-L1H2 13.26 20.27

Example 6: Humanized Anti-CSF1R Antibodies Inhibit Ligand-Induced CSF1RPhosphorylation

CSF1R is phosphorylated in the presence of ligands CSF1 or IL34. Thehumanized antibodies, along with their parental chimeric antibodies(cAbs), were tested for their ability to inhibit CSF1R phosphorylationinduced by either ligand, as follows.

Inhibition of CSF1-Induced Phosphorylation

CSF1R (SEQ ID NO: 2)-transfected CHO cells were incubated with serialdilutions of each humanized antibody or a parental chimeric antibody,beginning at 8 μg/ml, for 60 min on ice, after which 3.3 nM of humanCSF1 (M-CSF, R&D Systems) was added to the cells. (For the 0301 seriesof humanized antibodies, serial dilutions beginning at 2 μg/ml ofhumanized antibody and parental chimeric antibody was used.) The cellswere incubated for 3 minutes at 37° C., and then lysed by addition of1/10× volume of 10× cell lysis buffer (Cell Signaling Technology). Theamount of phosphorylated CSF1R in the cell lysates was quantified usinga human phospho-M-CSF R ELISA kit (R&D Systems) according to themanufacturer's instructions.

The results of that experiment are shown in FIGS. 6A to 6C. All of thehumanized antibodies were able to inhibit human CSF1-inducedphosphorylation of human CSF1R ECD within the range of concentrationstested.

Inhibition of IL34-Induced Phosphorylation

CSF1R (SEQ ID NO: 2)-transfected CHO cells were incubated with 0.002 to8 μg/ml of each humanized antibody or a parental chimeric antibody for60 min on ice, after which 3.3 nM of human IL34 (FivePrime Therapeutics;SEQ ID NO: 68) was added to the cells. The cells were incubated for 3minutes at 37° C., and then lysed by addition of 1/10× volume of 10×cell lysis buffer (Cell Signaling Technology). The amount ofphosphorylated CSF1R in the cell lysates was quantified using a humanphospho-M-CSF R ELISA kit (R&D Systems) according to the manufacturer'sinstructions.

The results of that experiment are shown in FIGS. 7A to 7C. All of thehumanized antibodies were able to inhibit human IL34-inducedphosphorylation of human CSF1R within the range of concentrationstested.

Example 7: Humanized Anti-CSF1R Antibodies Block Human CSF1 and HumanIL34 Binding to Human and Cynomolgous CSF1R Human CSF1/CSF1R BlockingActivity

The humanized antibodies, along with the parental chimeric antibodies(cAbs), were tested for their ability to block human CSF1 binding tohuman and cynomolgus CSF1R ECD, as follows.

Recombinant Human CSF1 (M-CSF; R&D Systems) was biotinylated using anNH₂-Biotin Labeling Kit (Dojindo Molecular Technologies). One hundred μlof 1 μg/ml biotinylated CSF1 in PBST/0.1% BSA was added to the wells ofReacti-Bind Streptavidin coated plates (Pierce) pre-blocked withSuperBlock blocking buffer (Pierce) according to the manufacturer'sinstructions. Fifty μl of 0.5× serial dilutions of the humanizedantibody or parental chimeric antibody, beginning with 2000 ng/ml, wasincubated with 50 ng/ml hCSF1R ECD.506-Fc (SEQ ID NO: 6; FivePrimeTherapeutics) or 50 ng/ml cynoCSF1R ECD-Fc (FivePrime Therapeutics, SEQID NO: 8, but without the 19 amino acid leader sequence) in 100 μlPBST/0.1% BSA for 90 min at RT, after which the admix was transferred toone or more wells of a ligand-coated plate. After 90 min at RT, wellswere washed with PBST, and a 1:5000 dilution of an Fc-fragment-specificperoxidase-conjugated goat anti-human IgG (Jackson Immuno Research) inPBST/0.1% BSA was added to each well. After incubation at RT for 60 min,wells were washed with PBST/0.1% BSA, and o-phenylenediaminedihydrochloride peroxidase substrate (Sigma) was added to each well.After incubation at RT for 30 min, A450 values of each well were readdirectly on a SpectraMaxPlus spectrophotometer with SoftMaxPro software(Molecular Devices).

The results of that experiment for cynomolgus CSF1R are shown in FIGS.8A to 8C. All of the humanized antibodies based on Fabs 0301 and 0311were able to block human CSF1 binding to cynomolgus CSF1R ECD within therange of concentrations tested. None of the humanized antibodies basedon Fab 0302 showed similar blocking activity in that experiment comparedto the blocking activity of cAb 0302.

Human IL34/CSF1R Blocking Activity

The humanized antibodies were tested for their ability to block humanIL34 binding to human CSF1R ECD. The blocking activity of each humanizedantibody was determined as described above for blocking of CSF1, exceptrecombinant human IL34 (FivePrime Therapeutics; SEQ ID NO: 68) wasbiotinylated using an NH2-Biotin Labeling Kit (Dojindo MolecularTechnologies), and then 100 μl of 1 μg/ml biotinylated recombinant IL34in PBST/0.1% BSA was added to the wells of Reacti-Bind Streptavidincoated plates (Pierce) pre-blocked with SuperBlock blocking buffer(Pierce) according to the manufacturer's instructions.

The results of that experiment for cynomolgus CSF1R are shown in FIGS.9A to 9C. All of the humanized antibodies based on Fabs 0301 and 0311were able to block human IL34 binding to cynomolgus CSF1R ECD within therange of concentrations tested. None of the humanized antibodies basedon Fab 0302 showed similar blocking activity in that experiment comparedto the blocking activity of cAb 0302.

Calculation of IC50s

Table 4 shows the IC50, calculated using the non-linear regression(curve-fit) analysis algorithm of the GraphPad Prism software (GraphPadSoftware), for inhibition of ligand-induced CSF1R phosphorylation byeach humanized antibody. Table 4 also shows the IC50, calculated usingthe non-linear regression (curve-fit) analysis algorithm of the GraphPadPrism software (GraphPad Software), for blocking of ligand binding toCSF1R ECD by each humanized antibody. Finally, Table 4 shows the numberof amino acids in the framework regions of the light and heavy chain ofeach humanized antibody that were back-mutated to the correspondingmouse amino acid residue. For example, humanized antibody h0301L1H1 hasone amino acid in a light chain framework region that was back-mutatedto the mouse amino acid, and one amino acid in the heavy chain frameworkregions that was back-mutated to the mouse amino acid. Referring toFIGS. 1 and 2, the back-mutated amino acid in the light chain frameworkis at position 1 in framework 1, and the back-mutated amino acid in theheavy chain is at position 71 in framework 3 according to Kabatnumbering (see FIG. 1B).

TABLE 4 Blocking activity of humanized anti-CSF1R antibodies Human HumanCSF1/ IL34/ Back- Human Human Human Human mutated CSF1R CSF1R CSF1/IL34/ mouse ECD ECD CynoCSF1R CynoCSF1R residues Humanized IC50 IC50 ECDIC50 ECD IC50 in FRs Antibody (ng/ml) (ng/ml) (ng/ml) (ng/ml) (L + H)cAb0301 307.2 312.2 22.01 29.53 h0301-L0H0 1031 433 27.64 35.92 0 + 0h0301-L0H1 778.1 452.6 27.45 36.43 0 + 1 h0301-L0H2 1317 480.9 28.0537.37 0 + 4 h0301-L1H0 6150 378 25.53 34.84 1 + 0 h0301-L1H1 814.2 384.431.07 42.41 1 + 1 h0301-L1H2 682.1 397.1 27.77 36.53 1 + 4 cAb0302 263.5350.8 33.09 49.38 h0302-L0H1 927.7 615 15.55 2.00E+12 0 + 2 h0302-L1H1742 363.7 60.49 676.4 1 + 2 h0302-L2H1 384 303.1 89827 509.1 3 + 2h0302-L0H2 438.2 474.2 none 248.1 0 + 5 h0302-L1H2 597.8 495.3 1085541.3 1 + 5 h0302-L2H2 354.4 240.1 837.6 278.7 3 + 5 cAb 0311 577 994.243.47 52.1 h0311-L0H1 291.3 343.2 32.47 50.4 0 + 2 h0311-L1H1 507.5667.4 24.68 53.69 2 + 2 h0311-L0H2 435.5 633.3 25.96 40.79 0 + 5h0311-L1H2 419 578.2 30.76 48.56 2 + 5

Example 8: Humanized Anti-CSF1R Antibody Binding Constants

The k_(a), k_(d), and K_(D) for binding to human CSF1R ECD wasdetermined for each of the humanized antibodies as follows.

Binding kinetics of anti-CSF1R humanized antibodies to CSF1R ECD wasdetermined using Biacore T100 Surface Plasmon Resonance (SPR) (GEHealthcare Life Sciences, Piscataway, N.Y.). Each of the humanizedanti-CSF1R antibodies was captured on a CM5 sensor chip immobilized withanti-Human IgG antibody using the Human antibody capture kit (GEHealthcare Life Sciences, Piscataway, N.Y.) at 150 RU so that the Rmaxvalue for hCSF1R ECD.506 (SEQ ID NO: 5) binding was 100 RU. Rmax valuesof less than 150 RU are recommended for accurately determining kineticvalues. 10 mM Hepes buffered saline, pH 7.4, with 0.05% Tween20 (HPS-P;GE Healthcare Life Sciences, Piscataway, N.Y.) was used as the runningand dilution buffer. hCSF1R ECD.506 was injected at six concentrations(90 nM, 30 nM, 10 nM, 3.33 nM, 1.11 nM, and 0 nM) for 2 minutes anddissociation was observed for 5 minutes to determine humanizedantibody/hCSF1R ECD binding kinetic parameters. The associationconstant, dissociation constant, affinity, and binding capacity of eachof the Fabs for human CSF1R ECD was calculated using the Biacore T100Evaluation software package using the 1:1 binding model.

The results of the kinetic determinations are shown in Table 5.

TABLE 5 Humanised antibody binding affinity for human CSF1R k_(a) K_(d)K_(D) huAbAb (M⁻¹s⁻¹) (s⁻¹) (nM) huAb 0301-L0H0 3.22 × 10⁶ 1.11 × 10−⁰³0.35 huAb 0301-L0H1 3.56 × 10⁶ 1.22 × 10−⁰³ 0.34 huAb 0301-L0H2 2.32 ×10⁶ 6.60 × 10−⁰⁴ 0.28 huAb 0301-L1H0 3.29 × 10⁶ 1.15 × 10−⁰³ 0.35 huAb0301-L1H1 2.87 × 10⁶ 9.21 × 10−⁰⁴ 0.32 huAb 0301-L1H2 2.95 × 10⁶ 7.42 ×10−⁰⁴ 0.25 huAb 0302-L0H1 3.54 × 10⁶ 3.69 × 10−⁰³ 1.04 huAb 0302-L1H13.47 × 10⁶ 4.04 × 10−⁰³ 1.17 huAb 0302-L2H1 1.60 × 10⁶ 9.14 × 10−⁰⁴ 0.57huAb 0302-L0H2 3.40 × 10⁶ 1.79 × 10−⁰³ 0.53 huAb 0302-L1H2 2.71 × 10⁶1.53 × 10−⁰³ 0.56 huAb 0302-L2H2 1.84 × 10⁶ 8.40 × 10−⁰⁴ 0.46 huAb0311-L0H1 1.22 × 10⁶ 5.40 × 10−⁰⁴ 0.44 huAb 0311-L1H1 1.32 × 10⁶ 6.64 ×10−⁰⁴ 0.50 huAb 0311-L0H2 1.34 × 10⁶ 4.73 × 10−⁰⁴ 0.35 huAb 0311-L1H21.51 × 10⁶ 6.09 × 10−⁰⁴ 0.40

All but two of the humanized antibodies showed sub-nanomolar bindingaffinities for human CSF1R ECD, and the remaining two humanizedantibodies showed binding affinities for human CSF1R ECD of less than 2nM.

Example 9: Humanized Anti-CSF1R Antibodies Block Ligand-InducedPhosphorylation

Based on the data above, including CSF1R binding and ligand inhibition,and the likelihood of immunogenicity for each humanized antibody, threehumanized antibodies were selected for further study: 0301-L0H0,0301-L1H0, and 0311-L0H1.

After confirming that 0301-L0H0, 0301-L1H0, and 0311-L0H1 each bind toCSF1R on the surface of cells (data not shown), each of the antibodieswas tested for the ability to block ligand-induced CSF1R phosphorylationin CHO cells, as described in Example 6.

The results of that experiment are shown in FIG. 10. All three of thehumanized antibodies tested blocked both CSF1-induced (A) andIL34-induced (B) phosphorylation of CSF1R in CHO cells. Table 6 showsthe IC50 for blocking of ligand-induced CSF1R phosphorylation for eachantibody.

TABLE 6 Ligand-induced phosphorylation blocking IC50 for humanizedantibodies CSF1 IL34 blocking blocking Humanized IC50 IC50 antibody(ng/ml) (ng/ml) 0301-L0H0 305.4 340.8 0301-L1H0 213.2 242.2 0311-L0H1127.2 337.6

Example 10: Humanized Anti-CSF1R Antibodies Block Ligand-InducedProliferation/Survival Responses of Primary Human Monocytes

Humanized antibodies 0301-L0H0, 0301-L1H0, and 0311-L0H1 were tested fortheir ability to block ligand-induced monocyte proliferation/survivalresponses as follows.

Human peripheral blood mononuclear cells (PBMCs) were isolated fromhealthy donor blood by centrifugation onto a Ficoll-Paque cushion (GEHealthcare Bio-Sciences) according to the manufacturer's instructions.Peripheral blood monocytes were subsequently isolated from the recoveredPBMC fraction by centrifugation onto a 48.5% Percoll™ cushion (GEHealthcare Bio-Sciences). After recovery from the Percoll™ cushion, thepurified peripheral blood monocytes were stimulated with 162 pMrecombinant human CSF1 or 1.6 nM recombinant human IL34 (both from R&DSystems) in the presence or absence of serial dilutions of humanisedantibody 0301-L0H0, humanised antibody 0301-L1H0, or humanised antibody0311-L0H1. After incubation at 37° C. for 48 hours, relative cellularATP content of each individual culture was assessed using CellTiter-Glo®reagent (Promega) according to the manufacturer's instructions. In thisassay, relative cellular ATP content is directly proportional to thenumber of viable cells in culture, and thus reflects monocyteproliferation/survival responses.

The results of that experiment are shown in FIG. 11. All three of thehumanized antibodies tested were able to block monocyteproliferation/survival responses following CSF1 (A) or IL34 (B)stimulation. Table 7 shows the IC50s for blocking of ligand-inducedmonocyte proliferation/survival responses for each antibody. The valuesshown in Table 7 represent the range observed from the three differentprimary donors tested.

TABLE 7 Monocyte proliferation/survival blocking IC50 for humanizedantibodies CSF1 IL34 blocking blocking Humanized IC50 IC50 antibody(ng/ml) (ng/ml) 0301-L0H0 31.9-77.5 12.2-29.9 0301-L1H0 19.0-71.910.5-30.6 0311-L0H1  75.9-134.8  26.9-152.2

Example 11: Humanized Anti-CSF1R Antibodies do not Directly StimulatePrimary Human Monocyte Proliferation or Survival Responses

Humanized antibodies 0301-L0H0, 0301-L1H0, and 0311-L0H1 were tested fortheir ability to directly stimulate primary monocyte proliferationand/or survival, as follows.

Human peripheral blood monocytes were isolated as described in Example10. Serial dilutions of humanised antibody 0301-L0H0, humanised antibody0301-L1H0, or humanised antibody 0311-L0H1 were added to the monocytesin the absence of stimulation either by exogenous CSF1 or by exogenousIL34. After incubation at 37° C. for 48 hours, relative ATP content ofeach individual culture was assessed using CellTiter Glo® reagent(Promega) as in Example 10. The experiment was carried out on peripheralblood monocytes from three different donors.

The results of that experiment are shown in FIG. 12. None of thehumanized antibodies stimulated primary monocyte proliferation orsurvival in either of the primary monocyte preparations tested.

Table of Sequences

Table 8 provides certain sequences discussed herein. All polypeptide andantibody sequences are shown without leader sequences, unless otherwiseindicated.

TABLE 8 Sequences and Descriptions SEQ ID NO Description Sequence 1hCSF1R IPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL(full-length, STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEno leader DQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFsequence) IQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHTHP PDEFLFTPVVVACMSIMALL LLLLLLLLYK YKQKPKYQVR WKIIESYEGN SYTFIDPTQLPYNEKWEFPR NNLQFGKTLG AGAFGKVVEA TAFGLGKEDA VLKVAVKMLKSTAHADEKEA LMSELKIMSH LGQHENIVNL LGACTHGGPV LVITEYCCYGDLLNFLRRKA EAMLGPSLSP GQDPEGGVDY KNIHLEKKYV RRDSGFSSQGVDTYVEMRPV STSSNDSFSE QDLDKEDGRP LELRDLLHFS SQVAQGMAFLASKNCIHRDV AARNVLLTNG HVAKIGDFGL ARDIMNDSNY IVKGNARLPVKWMAPESIFD CVYTVQSDVW SYGILLWEIF SLGLNPYPGI LVNSKFYKLVKDGYQMAQPA FAPKNIYSIM QACWALEPTH RPTFQQICSF LQEQAQEDRRERDYTNLPSS SRSGGSGSSS SELEEESSSE HLTCCEQGDI AQPLLQPNNY QFC 2 hCSF1RMGPGVLLLLL VATAWHGQGI PVIEPSVPEL VVKPGATVTL RCVGNGSVEW (full-length, +DGPPSPHWTL YSDGSSSILS TNNATFQNTG TYRCTEPGDP LGGSAAIHLY leaderVKDPARPWNV LAQEVVVFED QDALLPCLLT DPVLEAGVSL VRVRGRPLMR sequence)HTNYSFSPWH GFTIHRAKFI QSQDYQCSAL MGGRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASSVD VNFDVFLQHN NTKLAIPQQSDFHNNRYQKV LTLNLDQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLNLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTF INGSGTLLCA ASGYPQPNVT WLQCSGHTDR CDEAQVLQVWDDPYPEVLSQ EPFHKVTVQS LLTVETLEHN QTYECRAHNS VGSGSWAFIPISAGAHTHPP DEFLFTPVVV ACMSIMALLL LLLLLLLYKY KQKPKYQVRWKIIESYEGNS YTFIDPTQLP YNEKWEFPRN NLQFGKTLGA GAFGKVVEATAFGLGKEDAV LKVAVKMLKS TAHADEKEAL MSELKIMSHL GQHENIVNLLGACTHGGPVL VITEYCCYGD LLNFLRRKAE AMLGPSLSPG QDPEGGVDYKNIHLEKKYVR RDSGFSSQGV DTYVEMRPVS TSSNDSFSEQ DLDKEDGRPLELRDLLHFSS QVAQGMAFLA SKNCIHRDVA ARNVLLTNGH VAKIGDFGLARDIMNDSNYI VKGNARLPVK WMAPESIFDC VYTVQSDVWS YGILLWEIFSLGLNPYPGIL VNSKFYKLVK DGYQMAQPAF APKNIYSIMQ ACWALEPTHRPTFQQICSFL QEQAQEDRRE RDYTNLPSSS RSGGSGSSSS ELEEESSSEHLTCCEQGDIA QPLLQPNNYQ FC 5 hCSF1RIPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506STNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEDQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFIQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAH 6 hCSF1RIPVIEPSVPE LVVKPGATVT LRCVGNGSVE WDGPPSPHWT LYSDGSSSIL ECD.506-FcSTNNATFQNT GTYRCTEPGD PLGGSAAIHL YVKDPARPWN VLAQEVVVFEDQDALLPCLL TDPVLEAGVS LVRVRGRPLM RHTNYSFSPW HGFTIHRAKFIQSQDYQCSA LMGGRKVMSI SIRLKVQKVI PGPPALTLVP AELVRIRGEAAQIVCSASSV DVNFDVFLQH NNTKLAIPQQ SDFHNNRYQK VLTLNLDQVDFQHAGNYSCV ASNVQGKHST SMFFRVVESA YLNLSSEQNL IQEVTVGEGLNLKVMVEAYP GLQGFNWTYL GPFSDHQPEP KLANATTKDT YRHTFTLSLPRLKPSEAGRY SFLARNPGGW RALTFELTLR YPPEVSVIWT FINGSGTLLCAASGYPQPNV TWLQCSGHTD RCDEAQVLQV WDDPYPEVLS QEPFHKVTVQSLLTVETLEH NQTYECRAHN SVGSGSWAFI PISAGAHEPK SSDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK 7 cyno CSF1RMGPGVLLLLL VVTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW ECD (withDGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY leaderVKDPARPWNV LAKEVVVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR sequence)HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRSDFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVWVDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIP ISAGAR 8cyno CSF1R MGPGVLLLLL VVTAWHGQGI PVIEPSGPEL VVKPGETVTL RCVGNGSVEW ECD-FcDGPISPHWTL YSDGPSSVLT TTNATFQNTR TYRCTEPGDP LGGSAAIHLY (with leaderVKDPARPWNV LAKEVVVFED QDALLPCLLT DPVLEAGVSL VRLRGRPLLR sequence)HTNYSFSPWH GFTIHRAKFI QGQDYQCSAL MGSRKVMSIS IRLKVQKVIPGPPALTLVPA ELVRIRGEAA QIVCSASNID VDFDVFLQHN TTKLAIPQRSDFHDNRYQKV LTLSLGQVDF QHAGNYSCVA SNVQGKHSTS MFFRVVESAYLDLSSEQNLI QEVTVGEGLN LKVMVEAYPG LQGFNWTYLG PFSDHQPEPKLANATTKDTY RHTFTLSLPR LKPSEAGRYS FLARNPGGWR ALTFELTLRYPPEVSVIWTS INGSGTLLCA ASGYPQPNVT WLQCAGHTDR CDEAQVLQVWVDPHPEVLSQ EPFQKVTVQS LLTAETLEHN QTYECRAHNS VGSGSWAFIPISAGARGSEP KSSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTPEVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 3 Light chainMETDTLLLWV LLLWVPGSTG leader sequence 4 Heavy chain MAVLGLLLCL VTFPSCVLSleader sequence 9 Fab 0301EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD heavy chainINPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARES variablePYFSNLYVMD YWGQGTSVTV SS region 10 Fab 0301NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL light chainLIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLS variableTFGGGTKLEI K region 11 Fab 0302EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYF variableDGTFDYALDY WGQGTSITVS S region 12 Fab 0302DVVVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL light chainLIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPW variableTFGGGTRLEI K region 13 Fab 0311EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRAL variableYHSNFGWYFD SWGKGTTLTV SS region 14 Fab 0311DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL light chainLIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPW variableTFGGGTRLEI K region 15 0301 heavy GYTFTDNYMI chain CDR1 16 0301 heavyDINPYNGGTT FNQKFKG chain CDR2 17 0301 heavy ESPYFSNLYV MDY chain CDR3 180301 light KASQSVDYDG DNYMN chain CDR1 19 0301 light AASNLES chain CDR220 0301 light HLSNEDLST chain CDR3 21 0302 heavy GYTFSDFNIH chain CDR122 0302 heavy YINPYTDVTV YNEKFKG chain CDR2 23 0302 heavy YFDGTFDYAL DYchain CDR3 24 0302 light RASESVDNYG LSFMN chain CDR1 25 0302 lightTASNLES chain CDR2 26 0302 light QQSKELPWT chain CDR3 27 0311 heavyGYIFTDYNMH chain CDR1 28 0311 heavy EINPNNGVVV YNQKFKG chain CDR2 290311 heavy ALYHSNFGWY FDS chain CDR3 30 0311 light KASQSVDYDG DSHMNchain CDR1 31 0311 light TASNLES chain CDR2 32 0311 light QQGNEDPWTchain CDR3 33 cAb 0301EVQLQQSGPE LVRPGASVKM SCKASGYTFT DNYMIWVKQS HGKSLEWIGD heavy chainINPYNGGTTF NQKFKGKATL TVEKSSSTAY MQLNSLTSED SAVYYCARESPYFSNLYVMD YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 34 cAb 0301NIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDNYMNWY QQKPGQPPKL light chainLIYAASNLES GIPARFSGSG SGTDFTLNIH PVEEEDAATY YCHLSNEDLSTFGGGTKLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 35 cAb 0302EIQLQQSGPE LVKPGASVKM SCKASGYTFS DFNIHWVKQK PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGKATL TSDRSSSTAY MDLSSLTSED SAVYYCASYFDGTFDYALDY WGQGTSITVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 36 cAb 0302DVVVTQTPAS LAVSLGQRAT ISCRASESVD NYGLSFMNWF QQKPGQPPKL light chainLIYTASNLES GIPARFSGGG SRTDFTLTID PVEADDAATY FCQQSKELPWTFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 37 cAb 0311EIQLQQSGPD LMKPGASVKM SCKASGYIFT DYNMHWVKQN QGKSLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSSSTAY MDLHSLTSED SAVYYCTRALYHSNFGWYFD SWGKGTTLTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 38 cAb 0311DIVLTQSPAS LAVSLGQRAT ISCKASQSVD YDGDSHMNWY QQKPGQPPKL light chainLIYTASNLES GIPARFSGSG SGADFTLTIH PVEEEDAATY YCQQGNEDPWTFGGGTRLEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 39 h0301-H0QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 40 h0301-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 41 h0301-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD heavy chainINPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARES variablePYFSNLYVMD YWGQGTLVTV SS region 42 H0302-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY heavy chainINPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYF variableDGTFDYALDY WGQGTLVTVS S region 43 H0302-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYF variableDGTFDYALDY WGQGTLVTVS S region 44 H0311-H1QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRAL variableYHSNFGWYFD SWGQGTLVTV SS region 45 H0311-H2QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRAL variableYHSNFGWYFD SWGQGTLVTV SS region 46 H0301-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS variableTFGGGTKVEI K region 47 h0301-L1NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLS variableTFGGGTKVEI K region 48 H0302-L0EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 49 H0302-L1EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 50 H0302-L2EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPW variableTFGQGTKVEI K region 51 H0311-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPW variableTFGQGTKVEI K region 52 H0311-L1DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPW variableTFGQGTKVEI K region 53 h0301-H0QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 54 h0301-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWMGD heavy chainINPYNGGTTF NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 55 h0301-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFT DNYMIWVRQA PGQGLEWIGD heavy chainINPYNGGTTF NQKFKGRATL TVDKSTSTAY MELSSLRSED TAVYYCARESPYFSNLYVMD YWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 56 H0302-H1QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWMGY heavy chainINPYTDVTVY NEKFKGRVTI TSDKSTSTAY MELSSLRSED TAVYYCASYFDGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 57 H0302-H2QVQLVQSGAE VKKPGSSVKV SCKASGYTFS DFNIHWVRQA PGQGLEWIGY heavy chainINPYTDVTVY NEKFKGRATL TSDKSTSTAY MELSSLRSED TAVYYCASYFDGTFDYALDY WGQGTLVTVS SASTKGPSVF PLAPCSRSTS ESTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTKTYTCNVDHKP SNTKVDKRVE SKYGPPCPPC PAPEFLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVYTLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLDSDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK 58 H0311-H1QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGRVTI TVDKSTSTAY MELSSLRSED TAVYYCTRALYHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 59 H0311-H2QVQLVQSGAE VKKPGSSVKV SCKASGYIFT DYNMHWVRQA PGQGLEWMGE heavy chainINPNNGVVVY NQKFKGTTTL TVDKSTSTAY MELSSLRSED TAVYYCTRALYHSNFGWYFD SWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTKTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPKDTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQVYTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVLDSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 60 h0301-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLSTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 61 h0301-L1NIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDNYMNWY QQKPGQAPRL light chainLIYAASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCHLSNEDLSTFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 62 H0302-L0EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 63 H0302-L1EIVLTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 64 H0302-L2EIVVTQSPAT LSLSPGERAT LSCRASESVD NYGLSFMNWF QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SRTDFTLTIS SLEPEDFAVY YCQQSKELPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 65 H0311-L0EIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQQGNEDPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 66 H0311-L1DIVLTQSPAT LSLSPGERAT LSCKASQSVD YDGDSHMNWY QQKPGQAPRL light chainLIYTASNLES GIPARFSGSG SGADFTLTIS SLEPEDFAVY YCQQGNEDPWTFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKVQWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEVTHQGLSSPVT KSFNRGEC 67 HumanEEVSEYCSHM IGSGHLQSLQ RLIDSQMETS CQITFEFVDQ EQLKDPVCYL CSF1KKAFLLVQDI MEDTMRFRDN TPNAIAIVQL QELSLRLKSC FTKDYEEHDKACVRTFYETP LQLLEKVKNV FNETKNLLDK DWNIFSKNCN NSFAECSSQG HERQSEGS 68Human IL34 NEPLEMWPLT QNEECTVTGF LRDKLQYRSR LQYMKHYFPI NYKISVPYEGVFRIANVTRL QRAQVSEREL RYLWVLVSLSATESVQDVLL EGHPSWKYLQEVQTLLLNVQ QGLTDVEVSP KVESVLSLLN APGPNLKLVR PKALLDNCFRVMELLYCSCC KQSSVLNWQD CEVPSPQSCS PEPSLQYAAT QLYPPPPWSPSSPPHSTGSV RPVRAQGEGL LP 69 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor AFR1 70 Human WVRQAPGQGL EWMG acceptor A FR2 71 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor A FR3 72 Human WGQGTLVTVS Sacceptor A FR4 73 Human QVQLVQSGAE VKKPGSSVKV SCKAS acceptor B FR1 74Human WVRQAPGQGL EWMG acceptor B FR2 75 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor B FR3 76 Human WGQGTLVTVSSacceptor B FR4 77 Human acceptor C QVQLVQSGAE VKKPGSSVKV SCKAS FR1 78Human WVRQAPGQGL EWMG acceptor C FR2 79 HumanRVTITADKST STAYMELSSL RSEDTAVYYC AR acceptor C FR3 80 Human WGQGTLVTVS Sacceptor C FR4 81 Human EIVLTQSPAT LSLSPGERAT LSC acceptor D FR1 82Human WYQQKPGQAP RLLIY acceptor D FR2 83 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor D FR3 84 Human FGGGTKVEIKacceptor D FR4 85 Human EIVLTQSPAT LSLSPGERAT LSC acceptor E FR1 86Human WYQQKPGQAP RLLIY acceptor E FR2 87 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor E FR3 88 Human FGQGTKVEIKacceptor E FR4 89 Human EIVLTQSPAT LSLSPGERAT LSC acceptor F FR1 90Human WYQQKPGQAP RLLIY acceptor F FR2 91 HumanGIPARFSGSG SGTDFTLTIS SLEPEDFAVY YC acceptor F FR3 92 Human FGQGTKVEIKacceptor F FR4 93 mCSF1RAPVIEPSGPE LVVEPGETVT LRCVSNGSVE WDGPISPYWT LDPESPGSTL ECD-FcTTRNATFKNT GTYRCTELED PMAGSTTIHL YVKDPAHSWN LLAQEVTVVEGQEAVLPCLI TDPALKDSVS LMREGGRQVL RKTVYFFSPW RGFIIRKAKVLDSNTYVCKT MVNGRESTST GIWLKVNRVH PEPPQIKLEP SKLVRIRGEAAQIVCSATNA EVGFNVILKR GDTKLEIPLN SDFQDNYYKK VRALSLNAVDFQDAGIYSCV ASNDVGTRTA TMNFQVVESA YLNLTSEQSL LQEVSVGDSLILTVHADAYP SIQHYNWTYL GPFFEDQRKL EFITQRAIYR YTFKLFLNRVKASEAGQYFL MAQNKAGWNN LTFELTLRYP PEVSVTWMPV NGSDVLFCDVSGYPQPSVTW MECRGHTDRC DEAQALQVWN DTHPEVLSQK PFDKVIIQSQLPIGTLKHNM TYFCKTHNSV GNSSQYFRAV SLGQSKQEPK SSDKTHTCPPCPAPELLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWYVDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIAVEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVMHEALHNHYTQ KSLSLSPGK 94 HumanASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV IgG4 S241PHTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVESKYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYKCKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEGNVFSCSVMHE ALHNHYTQKS LSLSLGK 95 Human IgκRTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSGNSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

1.-83. (canceled)
 84. A conjugate comprising a cytotoxic agent and anantibody that binds to human colony stimulating factor 1 receptor(CSF1R); wherein the antibody comprises a heavy chain comprising a heavychain (HC) CDR1 having the sequence of SEQ ID NO:15, an HC CDR2 havingthe sequence of SEQ ID NO:16, and an HC CDR3 having the sequence of SEQID NO:17, and a light chain comprising a light chain (LC) CDR1 havingthe sequence of SEQ ID NO:18, a LC CDR2 having the sequence of SEQ IDNO:19, and a LC CDR3 having the sequence of SEQ ID NO:20.
 85. Theconjugate of claim 84, wherein the cytotoxic agent is a radioisotope,toxin, or chemotherapy agent.
 86. The conjugate of claim 84, wherein thecytotoxic agent is a chemotherapy agent.
 87. The conjugate of claim 84,wherein the conjugate further comprises a label.
 88. A conjugatecomprising a label and an antibody that binds to human colonystimulating factor 1 receptor (CSF1R); wherein the antibody comprises aheavy chain comprising a heavy chain (HC) CDR1 having the sequence ofSEQ ID NO:15, an HC CDR2 having the sequence of SEQ ID NO:16, and an HCCDR3 having the sequence of SEQ ID NO:17, and a light chain comprising alight chain (LC) CDR1 having the sequence of SEQ ID NO:18, a LC CDR2having the sequence of SEQ ID NO:19, and a LC CDR3 having the sequenceof SEQ ID NO:20.
 89. The conjugate of claim 88, wherein the labelfacilitates detection of the antibody.
 90. The conjugate of claim 88,wherein the label facilitates detection of human CSF1R.
 91. Theconjugate of claim 88, wherein the label is a radioisotope, fluorescentgroup, enzymatic group, chemiluminescent group, biotin, epitope tag, ormetal-binding tag.
 92. The conjugate of claim 84, wherein the antibodyblocks binding of human colony stimulating factor 1 (CSF-1) and humaninterleukin 34 (IL-34) to human CSF1R.
 93. The conjugate of claim 84,wherein the antibody heavy chain comprises the amino acid sequence ofSEQ ID NO: 39 and wherein the antibody light chain comprises the aminoacid sequence of SEQ ID NO:
 46. 94. The conjugate of claim 84, whereinthe antibody is an antigen binding fragment.
 95. The conjugate of claim94, wherein the antibody is a Fab, an Fv, an scFv, a Fab′, or a (Fab′)2.96. The conjugate of claim 84, wherein the antibody is an IgG.
 97. Theconjugate of claim 84, wherein the antibody is an IgG4 comprising anS241P mutation in at least one IgG4 heavy chain constant region.
 98. Theconjugate of claim 84, wherein the antibody comprises a full lengthheavy chain and/or a full length light chain.
 99. The conjugate of claim93, wherein the antibody is an antigen binding fragment.
 100. Theconjugate of claim 99, wherein the antibody is a Fab, an Fv, an scFv, aFab′, or a (Fab′)2.
 101. The conjugate of claim 93, wherein the antibodyis an IgG.
 102. The conjugate of claim 93, wherein the antibody is anIgG4 comprising an S241P mutation in at least one IgG4 heavy chainconstant region.
 103. The conjugate of claim 93, wherein the antibodycomprises a full length heavy chain and/or a full length light chain.104. The conjugate of claim 84, wherein the antibody heavy chaincomprises the amino acid sequence of SEQ ID NO: 53 and wherein theantibody light chain comprises the amino acid sequence of SEQ ID NO: 60.105. A pharmaceutical composition comprising the conjugate of claim 84and at least one pharmaceutically acceptable carrier.